Quinolinone-carboxamide compounds

ABSTRACT

The invention provides novel quinolinone-carboxamide 5-HT 4  receptor agonist compounds. The invention also provides pharmaceutical compositions comprising such compounds, methods of using such compounds to treat diseases associated with 5-HT 4  receptor activity, and processes and intermediates useful for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/267,720, filed Nov. 4, 2005 now U.S. Pat. No. 7,396,933, whichapplication claims the benefit of U.S. Provisional Application No.60/625,233, filed on Nov. 5, 2004, the entire disclosures of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to quinolinone-carboxamide compounds which areuseful as 5-HT₄ receptor agonists. The invention is also directed topharmaceutical compositions comprising such compounds, methods of usingsuch compounds for treating medical conditions mediated by 5-HT₄receptor activity, and processes and intermediates useful for preparingsuch compounds.

2. State of the Art

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter that iswidely distributed throughout the body, both in the central nervoussystem and in peripheral systems. At least seven subtypes of serotoninreceptors have been identified and the interaction of serotonin withthese different receptors is linked to a wide variety of physiologicalfunctions. There has been, therefore, substantial interest in developingtherapeutic agents that target specific 5-HT receptor subtypes.

In particular, characterization of 5-HT₄ receptors and identification ofpharmaceutical agents that interact with them has been the focus ofsignificant recent activity. (See, for example, the review by Langloisand Fischmeister, J. Med. Chem. 2003, 46, 319-344.) 5-HT₄ receptoragonists are useful for the treatment of disorders of reduced motilityof the gastrointestinal tract. Such disorders include irritable bowelsyndrome (IBS), chronic constipation, functional dyspepsia, delayedgastric emptying, gastroesophageal reflux disease (GERD), gastroparesis,post-operative ileus, intestinal pseudo-obstruction, and drug-induceddelayed transit. In addition, it has been suggested that some 5-HT₄receptor agonist compounds may be used in the treatment of centralnervous system disorders including cognitive disorders, behavioraldisorders, mood disorders, and disorders of control of autonomicfunction.

Despite the broad utility of pharmaceutical agents modulating 5-HT₄receptor activity, few 5-HT₄ receptor agonist compounds are in clinicaluse at present. Accordingly, there is a need for new 5-HT₄ receptoragonists that achieve their desired effects with minimal side effects.Preferred agents may possess, among other properties, improvedselectivity, potency, pharmacokinetic properties, and/or duration ofaction.

SUMMARY OF THE INVENTION

The invention provides novel compounds that possess 5-HT₄ receptoragonist activity. Among other properties, compounds of the inventionhave been found to be potent and selective 5-HT₄ receptor agonists.

Accordingly, the invention provides a compound of formula (I):

wherein:

R¹ is hydrogen, halo, hydroxy, C₁₋₄alkyl, or C₁₋₄alkoxy;

R² is C₃₋₄alkyl or C₃₋₆cycloalkyl;

R³ is hydroxy, C₁₋₃alkoxy, hydroxy-substituted C₁₋₄alkyl, or—OC(O)NR^(a)R^(b);

R⁴ is hydrogen or C₁₋₄alkyl;

X is selected from —N(R⁸)C(O)R⁹, —N(R⁸)S(O)₂R¹⁰, —S(R¹¹)O₂,—N(R⁸)C(O)OR¹², —N(R⁸)C(O)NR¹³R¹⁴, —N(R⁸)SO₂NR¹³R¹⁴, —C(O)NR¹³R¹⁴,—OC(O)NR¹³R¹⁴, —C(O)OR¹², —OR¹⁵, —NR⁸R¹⁶, cyano, —SR¹⁵, CF₃, pyridinyl,pyrrolyl, thiomorpholinyl, thiazolidinyl, 1,1-dioxoisothiazolidinyl,imidazolyl, indolyl, tetrahydrofuranyl, pyrrolidinyl and piperidinyl,wherein pyrrolidinyl is optionally substituted with oxo and piperidinylis optionally substituted with 1 to 3 halo;

R⁵ is hydrogen or C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substitutedwith hydroxy, C₁₋₃alkoxy, or cyano;

R⁶ and R⁷ are independently selected from hydrogen, hydroxy, halo, andC₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted with hydroxy orC₁₋₃alkoxy;

R⁸ is hydrogen or C₁₋₄alkyl;

or R⁵ and R⁸, R⁵ and R⁸, or R⁶ and R⁸ taken together form C₂₋₅alkylenyl,wherein C₂₋₅alkylenyl is optionally substituted with hydroxy, halo,hydroxy-substituted C₁₋₃alkyl, or C₁₋₃alkoxy;

or R³ and R⁵ taken together form —OCH₂CH₂—;

or R⁵ and R⁶ taken together form —(CH₂)_(q)-Q-(CH₂)_(q), wherein Q isoxygen or sulfur and q is independently 0, 1, or 2;

R⁹ is hydrogen, furanyl, or C₁₋₄alkyl, wherein C₁₋₄alkyl is optionallysubstituted with hydroxy or with from 1 to 3 halo;

R¹⁰ is hydrogen or C₁₋₄alkyl, wherein C₁₋₄alkyl is optionallysubstituted with —SO₂R^(c), C₃₋₆cycloalkyl or with from 1 to 3 halo;

or R⁸ and R¹⁰ taken together form C₃alkylenyl;

R¹¹ is hydrogen, C₁₋₄alkyl, or —NR^(b)R^(c);

or R⁵ and R¹¹ or R⁶ and R¹¹ taken together form C₂₋₅alkylenyl;

R¹², R¹³, and R¹⁴ are independently hydrogen or C₁₋₄alkyl;

R¹⁵ is hydrogen or C₁₋₄alkyl, wherein C₁₋₄alkyl is optionallysubstituted with hydroxy;

or R⁵ and R¹⁵ taken together form C₁₋₄alkylenyl;

R¹⁶ is —(CH₂)_(r)—R¹⁷, wherein r is 0, 1, 2, or 3;

R¹⁷ is selected from hydrogen, hydroxy, C₁₋₃alkyl, C₁₋₃alkoxy,—C(O)NR^(a)R^(b), —C(O)-morpholinyl, pyridinyl, pyrrolyl, morpholinyl,and tetrahydrofuranyl, wherein C₁₋₃alkoxy is optionally substituted withhydroxy;

R^(a), R^(b), and R^(c) are independently hydrogen or C₁₋₃alkyl; and

n is 1, 2, 3, or 4;

provided that when n is 1, X forms a carbon-carbon bond with the carbonatom bearing the substituents R⁶ and R⁷;

or a pharmaceutically-acceptable salt or solvate or stereoisomerthereof.

The invention also provides a pharmaceutical composition comprising atherapeutically effective amount of a compound of the invention and apharmaceutically-acceptable carrier.

The invention also provides a method of treating a disease or conditionassociated with 5-HT₄ receptor activity, e.g. a disorder of reducedmotility of the gastrointestinal tract, the method comprisingadministering to the mammal, a therapeutically effective amount of apharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a compound of the invention.

Further, the invention provides a method of treating a disease orcondition associated with 5-HT₄ receptor activity in a mammal, themethod comprising administering to the mammal, a therapeuticallyeffective amount of a pharmaceutical composition of the invention.

The compounds of the invention can also be used as research tools, i.e.to study biological systems or samples, or for studying the activity ofother chemical compounds. Accordingly, in another of its method aspects,the invention provides a method of using a compound of formula (I), or apharmaceutically acceptable salt or solvate or stereoisomer thereof, asa research tool for studying a biological system or sample or fordiscovering new 5-HT₄ receptor agonists, the method comprisingcontacting a biological system or sample with a compound of theinvention and determining the effects caused by the compound on thebiological system or sample.

In separate and distinct aspects, the invention also provides syntheticprocesses and intermediates described herein, which are useful forpreparing compounds of the invention.

The invention also provides a compound of the invention as describedherein for use in medical therapy, as well as the use of a compound ofthe invention in the manufacture of a formulation or medicament fortreating a disease or condition associated with 5-HT₄ receptor activity,e.g. a disorder of reduced motility of the gastrointestinal tract, in amammal.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides novel quinolinone-carboxamide 5-HT₄ receptoragonists of formula (I), or pharmaceutically-acceptable salts orsolvates or stereoisomers thereof. The following substituents and valuesare intended to provide representative examples of various aspects ofthis invention. These representative values are intended to furtherdefine such aspects and are not intended to exclude other values orlimit the scope of the invention.

In a specific aspect of the invention, R¹ is hydrogen, halo, orC₁₋₄alkyl.

In another specific aspect, R¹ is hydrogen, halo, or C₁₋₃alkyl.

In other specific aspects, R¹ is hydrogen, fluoro, chloro, bromo, ormethyl; or R¹ is hydrogen.

In a specific aspect, R² is C₃₋₄alkyl.

Representative R² groups include n-propyl, isopropyl, n-butyl,sec-butyl, and tert-butyl.

In another specific aspect, R² is isopropyl.

In a specific aspect, R³ is hydroxy, C₁₋₃alkoxy, hydroxy-substitutedC₁₋₂alkyl or —OC(O)NR^(a)R^(b). Representative R³ groups include, butare not limited to, hydroxy, methoxy, hydroxymethyl, 2-hydroxyethyl, and—OC(O)NR^(a)R^(b), wherein R^(a) and R^(b) are independently hydrogen ormethyl.

In other specific aspects, R³ is hydroxy, methoxy, hydroxymethyl,—OC(O)NHCH₃, or —OC(O)N(CH₃)₂; R³ is hydroxy or —OC(O)NHCH₃; or R³ ishydroxy.

In specific aspects, R⁴ is hydrogen or methyl; or R⁴ is hydrogen.

In a specific aspect, R⁵ is hydrogen or C₁₋₄alkyl, wherein C₁₋₄alkyl isoptionally substituted with hydroxy, C₁₋₃alkoxy, or cyano.

In another specific aspect, R⁵ is hydrogen, C₁₋₃alkyl, or C₁₋₃alkylsubstituted at the terminal position with hydroxy, C₁₋₃alkoxy, or cyano.Representative R⁵ groups include, but are not limited to, hydrogen,methyl, ethyl, 2-hydroxyethyl, 2-methoxyethyl, cyanomethyl, and2-cyanoethyl.

In another specific aspect, R⁵ is hydrogen, C₁₋₃alkyl, or C₁₋₃alkylsubstituted at the terminal position with hydroxy.

In a specific aspect, R⁶ and R⁷ are independently selected fromhydrogen, hydroxy, halo, and C₁₋₄alkyl, wherein C₁₋₄alkyl is optionallysubstituted with hydroxy or C₁₋₃alkoxy.

In another specific aspect, R⁶ and R⁷ are independently hydrogen,hydroxy, halo, C₁₋₂alkyl, or hydroxy-substituted C₁₋₂alkyl.Representative R⁶ and R⁷ groups include, but are not limited to,hydrogen, hydroxy, fluoro, chloro, hydroxyethyl, and hydroxymethyl.

In another specific aspect, R⁶ and R⁷ are each hydrogen.

In a specific aspect, R³ and R⁵ taken together form —OCH₂CH₂—.

In a specific aspect, n is 2 or 3 and R⁵ and R⁶ taken together form—CH₂CH₂O— or —CH₂CH₂OCH₂—.

In other specific aspects, n is 2 and R⁵ and R⁶ taken together formC₂₋₃alkylenyl or C₂alkylenyl.

In a specific aspect, X is selected from —N(R⁸)C(O)R⁹, —N(R⁸)S(O)₂R¹⁰,—S(R¹¹)O₂, —N(R⁸)C(O)OR¹², —N(R⁸)C(O)NR¹³R¹⁴, —N(R⁸)SO₂NR¹³R¹⁴,—C(O)NR¹³R¹⁴, —OC(O)NR¹³R¹⁴, —C(O)OR¹², —OR¹⁵, and cyano.

In another specific aspect, X is selected from —N(R⁸)C(O)R⁹;—N(R⁸)S(O)₂R¹⁰, —S(R¹¹)O₂, —N(R⁸)C(O)NR¹³R¹⁴, —C(O)NR¹³R¹⁴,—OC(O)NR¹³R¹⁴, —OR¹⁵, and cyano.

In yet another specific aspect, X is selected from —N(R⁸)C(O)R⁹,—N(R⁸)S(O)₂R¹⁰, —S(R¹¹)O₂, and —N(R⁸)C(O)NR¹³R¹⁴. In still anotherspecific aspect, X is —N(R⁸)S(O)₂R¹⁰.

In specific aspects, R⁸ is hydrogen or C₁₋₄alkyl; R⁸ is hydrogen orC₁₋₃alkyl; or R⁸ is hydrogen or methyl.

In a specific aspect, n is 2 and R⁵ and R⁸ taken together formC₂alkylenyl.

In another specific aspect, n is 2 and R⁵ and R⁸ taken together formC₃alkylenyl.

In a specific aspect, n is 2 and R⁶ and R⁸ taken together formC₂₋₃alkylenyl.

In a specific aspect, R⁹ is hydrogen, furanyl, or C₁₋₃alkyl, whereinC₁₋₃alkyl is optionally substituted with hydroxy. Representative R⁹groups include, but are not limited to, hydrogen, furanyl, methyl,ethyl, propyl, isopropyl, and 1-hydroxyethyl.

In other specific aspects, R⁹ is hydrogen or C₁₋₃alkyl; or R⁹ ishydrogen or methyl.

In a specific aspect, R¹⁰ is hydrogen or C₁₋₄alkyl, wherein C₁₋₄alkyl isoptionally substituted with —SO₂R^(c), C₃₋₆cycloalkyl or with from 1 to3 halo.

In another specific aspect, R¹⁰ is hydrogen or C₁₋₃alkyl, whereinC₁₋₃alkyl is optionally substituted with —SO₂R^(c) wherein R^(c) isC₁₋₃alkyl. Representative R¹⁰ groups include, but are not limited to,hydrogen, methyl, ethyl, propyl, isopropyl, and methanesulfonylmethyl.

In other specific aspects, R¹⁰ is hydrogen, C₁₋₃alkyl, ormethanesulfonylmethyl; or R¹⁰ is methyl, isopropyl, ormethanesulfonylmethyl; or R¹⁰ is methyl.

In specific aspects, R¹¹ is hydrogen, C₁₋₄alkyl, or —NR^(b)R^(c); R¹¹ ishydrogen or C₁₋₃alkyl; or R¹¹ is methyl.

In another specific aspect, n is 2 and R⁵ and R¹¹ taken together formC₂alkylenyl.

In yet another specific aspect, n is 2 or 3 and R⁶ and R¹¹ takentogether form C₂alkylenyl.

In specific aspects, R¹², R¹³, and R¹⁴ are independently hydrogen orC₁₋₃alkyl, or R¹², R¹³, and R¹⁴ are independently hydrogen or methyl.

In a specific aspect, R¹⁵ is hydrogen or C₁₋₄alkyl, wherein C₁₋₄alkyl isoptionally substituted with hydroxy.

In other specific aspects, R¹⁵ is hydrogen, C₁₋₃alkyl, or C₁₋₃alkylsubstituted at the terminal position with hydroxy; or R¹⁵ is hydrogen orC₁₋₃alkyl. Representative R¹⁵ groups include, but are not limited to,hydrogen, methyl, ethyl, and 2-hydroxyethyl.

In another specific aspect, R¹⁵ is hydrogen or methyl.

In a specific aspect, R¹⁶ is —(CH₂)_(r)—R¹⁷, wherein r is 0, 1, or 2 andR¹⁷ is selected from hydroxy, C₁₋₂alkoxy, —C(O)NR^(a)R^(b),—C(O)-morpholinyl, pyridinyl, morpholinyl, and tetrahydrofuranyl,wherein C₁₋₂alkoxy is optionally substituted at the terminal positionwith hydroxy and R^(a) and R^(b) are independently hydrogen or methyl.

In a specific aspect, n is 1, 2, or 3.

In another specific aspect, n is 2, 3, or 4.

In another specific aspect, n is 2 or 3.

In yet another specific aspect, n is 2

In one aspect, the invention provides a compound of formula (III):

wherein:

R¹ is hydrogen, halo, or C₁₋₃alkyl;

R² is C₃₋₄alkyl;

R³ is hydroxy, C₁₋₃alkoxy, hydroxy-substituted C₁₋₂alkyl, or—OC(O)NR^(a)R^(b);

R⁵ is hydrogen, C₁₋₃alkyl, or C₁₋₃alkyl substituted at the terminalposition with hydroxy or cyano;

R⁶ is hydrogen;

X is selected from —N(R⁸)C(O)R⁹; —N(R⁸)S(O)₂R¹⁰, —S(R¹¹)O₂,—N(R⁸)C(O)NR¹³R¹⁴, —C(O)NR¹³R¹⁴, —OC(O)NR¹³R¹⁴, —OR¹⁵, and cyano;

R⁸ is hydrogen or C₁₋₃alkyl;

R⁹ is hydrogen or C₁₋₃alkyl;

R¹⁰ is hydrogen or C₁₋₃alkyl, wherein C₁₋₃alkyl is optionallysubstituted with —SO₂R^(c) wherein R^(c) is C₁₋₃alkyl;

R¹³, R¹⁴, and R¹⁵ are independently hydrogen or C₁₋₃alkyl;

or R⁵ and R⁸, R⁵ and R⁶, or R⁵ and R¹¹ taken together form C₂alkylenyl;or

a pharmaceutically-acceptable salt or solvate or stereoisomer thereof.

In another aspect, the invention provides a compound of formula (II)wherein:

R¹ is hydrogen;

R² is C₃₋₄alkyl;

R³ is hydroxy, methoxy, hydroxymethyl, —OC(O)N(H)CH₃, or —OC(O)N(CH₃)₂;

R⁶ is hydrogen;

X is selected from —N(R⁸)C(O)R⁹; —N(R⁸)S(O)₂R¹⁰, —S(R¹¹)O₂, and—N(R⁸)C(O)NR¹³R¹⁴;

R⁵ and R⁸ taken together form C₂alkylenyl;

R⁹ is hydrogen or C₁₋₃alkyl;

R¹⁰ is hydrogen, C₁₋₃alkyl, or methanesulfonylmethyl;

R⁵ and R¹¹ taken together form C₂alkylenyl; and

R¹³ and R¹⁴ are independently hydrogen or C₁₋₃alkyl.

In separate aspects, the invention further provides compounds of formula(III):

wherein R¹, R², R³, and R⁹ take any of the values defined above;compounds of formula (IV):

wherein R¹, R², R³, and R¹⁰ take any of the values defined above;and compounds of formula (V):

wherein R¹, R², and R³ take any of the values defined above.

In still other specific aspects, the invention provides the compoundslisted in Tables I to XXVIII below.

The chemical naming conventions used herein are illustrated for thecompound of Example 1

which is designated 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid{(1S,3R,5R)-8-[2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}-amide,according to the AutoNom software, provided by MDL Information Systems,GmbH (Frankfurt, Germany). The designation (1S,3R,5R) describes therelative orientation of the bonds associated with the bicyclic ringsystem that are depicted as solid and dashed wedges. The compound isalternatively denoted asN-[(3-endo)-8-[2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl]-1-(1-methylethyl)-2-oxo-1,2-dihydro-3-quinolinecarboxamide.In all of the compounds of the invention listed explicitly below, thequinolinone-carboxamide group is endo to the azabicyclooctane group.

Particular mention may be made of the following compounds:

-   1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid    {(1S,3R,5R)-8-[2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;-   1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid    ((1S,3R,5R)-8-{2-hydroxy-3-[4-(propane-2-sulfonyl)piperazin-1-yl]propyl}-8-azabicyclo[3.2.1]oct-3-yl)-amide;-   1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid    {(1S,3R,5R)-8-[3-(1,1-dioxo-1λ⁶-thiomorpholin-4-yl)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;-   1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid    {(1S,3R,5R)-8-[(S)-2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;-   1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid    {(1S,3R,5R)-8-[(R)-2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;-   1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid    {(1S,3R,5R)-8-[3-(4-methanesulfonylmethanesulfonylpiperazin-1-yl)-2-methoxypropyl]-8-azabicyclo[3.2.1]-oct-3-yl}amide;-   1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid    {(1S,3R,5R)-8-[3-(4-acetylpiperazin-1-yl)-2-methoxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;-   1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid    {(1S,3R,5R)-8-[3-(1,1-dioxo-1λ⁶-thiomorpholin-4-yl)-2-methoxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;-   methylcarbamic acid    2-{(1S,3R,5R)-3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}-1-(4-methanesulfonylpiperazin-1-ylmethyl)ethyl    ester;-   methylcarbamic acid    1-(4-dimethylcarbamoylpiperazin-1-ylmethyl)-2-{(1S,3R,5R)-3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}ethyl    ester;-   methylcarbamic acid    1-[3-(acetylmethylamino)pyrrolidin-1-ylmethyl]-2-{(1S,3R,5R)-3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}ethyl    ester;-   methylcarbamic acid    1-(4-acetylpiperazin-1-ylmethyl)-2-{(1S,3R,5R)-3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}-ethyl    ester,-   methylcarbamic acid    (R)-2-{(1S,3R,5R)-3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}-1-(4-methanesulfonyl-piperazin-1-ylmethyl)ethyl    ester; and-   1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid    {(1S,3R,5R)-8-[3-hydroxy-2-(4-methanesulfonylpiperazin-1-ylmethyl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}-amide.

As exemplified by particular compounds listed above, the compounds ofthe invention may contain one or more chiral centers, in particular, thecompounds may contain a chiral center at the carbon atom in formulas (I)to (IV) bearing the substituent R³. Accordingly, the invention includesracemic mixtures, pure stereoisomers, and stereoisomer-enriched mixturesof such isomers, unless otherwise indicated. When a particularstereoisomer is shown, it will be understood by those skilled in theart, that minor amounts of other stereoisomers may be present in thecompositions of the invention unless otherwise indicated, provided thatany utility of the composition as a whole is not eliminated by thepresence of such other isomers.

Definitions

When describing the compounds, compositions and methods of theinvention, the following terms have the following meanings, unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Unless otherwisedefined, such alkyl groups typically contain from 1 to 10 carbon atoms.Representative alkyl groups include, by way of example, methyl, ethyl,n-propyl (n-Pr), isopropyl (i-Pr), n-butyl (n-Bu), sec-butyl, isobutyl,tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl andthe like.

The term “alkylenyl” means a divalent saturated hydrocarbon group whichmay be linear or branched or combinations thereof. Unless otherwisedefined, such alkylenyl groups typically contain from 1 to 10 carbonatoms. Representative alkylenyl groups include, by way of example,methylene, ethylene, n-propylene, n-butylene, propane-1,2-diyl(1-methylethylene), 2-methylpropane-1,2-diyl (1,1-dimethylethylene) andthe like.

The term “alkoxy” means the monovalent group -O-alkyl, where alkyl isdefined as above. Representative alkoxy groups include, by way ofexample, methoxy, ethoxy, propoxy, butoxy, and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclic groupwhich may be monocyclic or multicyclic. Unless otherwise defined, suchcycloalkyl groups typically contain from 3 to 10 carbon atoms.Representative cycloalkyl groups include, by way of example,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, and the like.

The term “halo” means fluoro, chloro, bromo or iodo.

The term “compound” means a compound that was synthetically prepared orprepared in any other way, such as by metabolism.

The term “therapeutically effective amount” refers to an amountsufficient to effect treatment when administered to a patient in need oftreatment.

The term “treatment” as used herein refers to the treatment of adisease, disorder, or medical condition in a patient, such as a mammal(particularly a human) which includes:

-   -   (a) preventing the disease, disorder, or medical condition from        occurring, i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease, disorder, or medical condition,        i.e., eliminating or causing regression of the disease,        disorder, or medical condition in a patient;    -   (c) suppressing the disease, disorder, or medical condition,        i.e., slowing or arresting the development of the disease,        disorder, or medical condition in a patient; or    -   (d) alleviating the symptoms of the disease, disorder, or        medical condition in a patient.

The term “pharmaceutically-acceptable salt” refers to a salt preparedfrom an acid or base which is acceptable for administration to apatient, such as a mammal. Such salts can be derived frompharmaceutically-acceptable inorganic or organic acids and frompharmaceutically-acceptable bases. Typically,pharmaceutically-acceptable salts of compounds of the present inventionare prepared from acids.

Salts derived from pharmaceutically-acceptable acids include, but arenot limited to, acetic, benzenesulfonic, benzoic, camphorsulfonic,citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic,hydrochloric, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pantothenic, phosphoric, succinic, sulfuric, tartaric,p-toluenesulfonic, xinafoic (1-hydroxy-2-naphthoic acid),napthalene-1,5-disulfonic acid and the like.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a compound of the invention or apharmaceutically-acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include by way of example, water, methanol, ethanol,isopropanol, acetic acid, and the like. When the solvent is water, thesolvate formed is a hydrate.

It will be appreciated that the term “or a pharmaceutically-acceptablesalt or solvate of stereoisomer thereof” is intended to include allpermutations of salts, solvates and stereoisomers, such as a solvate ofa pharmaceutically-acceptable salt of a stereoisomer of a compound offormula (I).

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino nitrogen. Representativeamino-protecting groups include, but are not limited to, formyl; acylgroups, for example alkanoyl groups, such as acetyl; alkoxycarbonylgroups, such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups,such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc);arylmethyl groups, such as benzyl (Bn), trityl (Tr), and1,1-di-(4′-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl(TMS) and tert-butyldimethylsilyl (TBDMS); and the like.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods and procedures.Although a particular aspect of the present invention is illustrated inthe schemes below, those skilled in the art will recognize that allaspects of the present invention can be prepared using the methodsdescribed herein or by using other methods, reagents and startingmaterials known to those skilled in the art. It will also be appreciatedthat where typical or preferred process conditions (i.e., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group, as well assuitable conditions for protection and deprotection, are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

In one method of synthesis, compounds of formula (I) in which R³ isdefined as hydroxy or hydroxy-substituted C₁₋₄alkyl are prepared asillustrated in Scheme A. (The substituents and variables shown in thefollowing schemes have the definitions provided herein unless otherwiseindicated).

In Scheme A, intermediate 2 is a secondary amine, and L represents aleaving group such as chloro, bromo, iodo, methanesulfonyl,p-toluenesulfonyl, or trifluoromethanesulfonyl.

For convenience, the moiety:

is represented by Y.

The reaction is typically conducted by contacting intermediate 1 withbetween about 1 and about 3 equivalents, each, of intermediates 2 and 3in an inert diluent, such as methanol or ethanol, in the presence of anexcess of base, for example between about 3 and about 6 equivalents, ofbase, such as N,N-diisopropylethylamine. The reaction is typicallyconducted at a temperature in the range of about 50° C. to about 80° C.for about 12 hours to about 24 hours, or until the reaction issubstantially complete. Optionally, equal molar equivalents ofintermediates 2 and 3 can be added in portions, as described in Example1, below.

The product of formula (I) is isolated and purified by conventionalprocedures. For example, the product can be concentrated to drynessunder reduced pressure, taken up in an aqueous weak acid solution andpurified by HPLC chromatography.

It will be understood that in the process of Scheme A and in otherprocesses described below using intermediate 1, intermediate 1 can besupplied in the form of the freebase or in a salt form, with appropriateadjustment of reaction conditions, as necessary, as known to thoseskilled in the art.

In Scheme A, the reaction of intermediate 1 with intermediates 2 and 3is accomplished in a single step. Alternatively, the reaction can beperformed in a stepwise manner. Using similar reaction conditions tothose described above, intermediates 1 and 3 can first be coupled toform an intermediate 5:

which is then reacted with the amine H—Y to provide a compound offormula (I). Alternatively, the amine can first be coupled tointermediate 3 to form an intermediate 10:

which is subsequently reacted with the quinolinone carboxamide-tropaneintermediate 1.

Compounds of formula (I) can also be prepared by N-alkylating a compoundof the form of formula (I) in which R² is defined as hydrogen, which canbe prepared according to Scheme A. The N-alkylation reaction istypically conducted by contacting a compound of formula (I) in which R²is hydrogen with between about 1 and about 4 equivalents of a compoundof the formula L′-R² in which L′ is a leaving group such as iodo orbromo. This reaction is typically conducted in a polar aprotic solventsuch as dimethylformamide in the presence of between about 2 and about 4equivalents of strong base, such as potassium tert-butoxide or sodiumhydride. Typically, the reaction is performed at a temperature ofbetween about 60° C. and about 100° C. for between about 6 and about 24hours, or until the reaction is substantially complete.

In yet another alternative, compounds of formula (I) in which R¹ isother than hydrogen are prepared by conventional processes fromcompounds of formula (I) in which R¹ is hydrogen.

In another method of synthesis, compounds of formula (I) in which R³ ishydroxy, C₁₋₃alkoxy, or —OC(O)NR^(a)R^(b) and the carbon atom bearingthe substituent R³ is not chiral can be prepared from an azetidineintermediate 11, as illustrated in Scheme B:

where L′ is a halo anion, such as Cl⁻ or Br⁻.

The reaction is typically conducted by contacting intermediate 11 withbetween about 1 and about 4 equivalents of intermediate 2 in an inertdiluent, such as ethanol, methanol, or dimethylformamide, in thepresence of an excess of base, for example between about 2 and about 4equivalents, of base, such as N,N-diisopropylethylamine,1,8-diazabicylco[5.4.0]undec-7-ene (DBU) or triethylamine. The reactionis typically conducted at a temperature in the range of about 50° C. toabout 80° C. for about 1 hour to about 6 hours, or until the reaction issubstantially complete. The product is isolated and purified byconventional means.

In yet another method of synthesis, compounds of formula (I) in which R³is hydroxy can be prepared as illustrated in Scheme C.

When an intermediate of formula 12 in which the carbon indicated by anasterisk is chiral is employed, the reaction of Scheme C is useful toprepare compounds of formula (I) having a chiral center at the carbonbearing the substituent R³. Typically, in the reaction of Scheme C,intermediate 1 is contacted with between about 1 and about 1.2equivalents of the epoxide 12 in an inert diluent such as ethanol ortoluene. The reaction is typically conducted at temperature in the rangeof about 50° C. to about 100° C. for about 12 hours to about 24 hours,or until the reaction is substantially complete. The product is isolatedand purified by conventional means.

The intermediates employed in Schemes A, B, and C above are preparedfrom readily available starting materials. For example, when R³ ishydroxy, an azetidine intermediate of formula 13 is prepared by theprocedure illustrated in Scheme D:

where L′ represents a halo leaving group, such as bromo, chloro, oriodo.

An intermediate of formula 1 is reacted with an oxirane compound,preferably 2-bromomethyloxirane (commonly, epibromohydrin) to form theazetidine salt of formula 13. This reaction is typically conducted bycontacting 1 with between about 2 and about 4 equivalents of2-bromomethyloxirane in a polar diluent, such as ethanol. The reactionis typically conducted at ambient temperature for between about 24 andabout 48 hours or until the reaction is substantially complete.

To form the azetidine intermediate 11 in which R³ is C₁₋₃alkoxy, theintermediate of formula 13 above is contacted with from slightly lessthan one equivalent to about one equivalent of a C₁₋₃alkylhalide in aninert diluent in the presence of between about 1 and about 3 equivalentsof a strong base, such as potassium tert-butoxide or sodium hydride. Thereaction is typically conducted at ambient temperature for between abouta quarter hour to an hour, or until the reaction is substantiallycomplete. Suitable inert diluents include dichloromethane,tetrahydrofuran, toluene, dimethylformamide, and the like.

The azetidine intermediate 11 in which R³ is a carbamic acid moiety ofthe form —OC(O)NR^(a)R^(b) can also be prepared from the intermediate offormula 13 in which R³ is hydroxy. For example, to prepare a compound offormula 11 in which R³ is —OC(O)N(H)CH₃ or —OC(O)N(CH₃)₂, intermediate13 is contacted with between about 1 and about 3 equivalents ofmethylisocyanate or dimethylisocyanate, respectively, in an inertdiluent in the presence of between about 1 and about 3 equivalents ofbase, such as N,N-diisopropylethylamine, and of a catalytic amount of astrong base such as potassium tert-butoxide or sodium hydride. Thereaction is typically conducted at ambient temperature for between about4 hours and about 24 hours, or until the reaction is substantiallycomplete.

A process for preparing intermediates of formula 1 is shown in Scheme E:

where P¹ represents an amino-protecting group. The protectedaminoazabicyclooctane, or commonly, aminotropane 15 is first reactedwith the substituted quinolinone carboxylic acid 14. Typically, thisreaction is conducted by first converting 14 to an acid chloride bycontacting 14 with at least one equivalent, preferably between about 1and about 2 equivalents of an activating agent, such as thionyl chlorideor oxalyl chloride in an aromatic diluent, such as toluene, benzene,xylene, or the like. The reaction is typically conducted at atemperature ranging from about 80° C. to about 120° C. for about 15minutes to about 4 hours, or until the reaction is substantiallycomplete.

The acid chloride solution is typically added to a biphasic mixture ofabout 1 equivalent of the aminotropane 15 to form a protectedintermediate, which is extracted by standard procedures. The biphasicmixture of 15 is generally prepared by dissolving 15 in an aromaticdiluent, such as used above, and adding an aqueous solution containingan excess of base, such as sodium hydroxide or potassium hydroxide,preferably about 2 to 5 equivalents of base.

Alternatively, the amide coupling of intermediate 15 with the carboxylicacid 14 can be performed in the presence of a coupling agent such as 1,3dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), orbenzotriazol-1-yloxytripyrrolidino-phosphonium hexafluorophosphate(PyBop), optionally combined with 1-hydroxy-7-azabenzotriazole (HOAt).In yet another alternative, the amide coupling of intermediate 15 withthe carboxylic acid 14 can be performed by converting 14 to an activatedester.

The protecting group P¹ is removed by standard procedures to provide anintermediate of formula 1. For example when the protecting group is Boc,typically removal is by treatment with an acid, such as trifluoroaceticacid, providing the acid salt of the intermediate. The acid salt ofintermediate 1 can be converted to the free base, if desired, byconventional treatment with base. The protecting group Cbz, for anotherexample, is conveniently removed by hydrogenolysis over a suitable metalcatalyst such as palladium on carbon.

The protected aminotropane 15 employed in the reactions described inthis application is prepared from readily available starting materials.For example, when the protecting group P¹ is Boc, the protectedaminotropane 16 is prepared by the procedure illustrated in Scheme F.

As described in detail in Example 1a below, to prepare the protectedintermediate 16, first, 2,5-dimethoxy tetrahydrofuran 17 is contactedwith between about 1 and 2 equivalents, preferably about 1.5 equivalentsof benzyl amine and a slight excess, for example about 1.1 equivalents,of 1,3-acetonedicarboxylic acid 18 in an acidic aqueous solution in thepresence of a buffering agent such as sodium hydrogen phosphate. Thereaction mixture is heated to between about 60° C. and about 100° C. toensure decarboxylation of any carboxylated intermediates in the product,8-benzyl-8-azabicyclo[3.2.1]octan-3-one 19, commonly N-benzyltropanone.

The intermediate 19 is typically reacted with a slight excess ofdi-tert-butyl dicarbonate (commonly (Boc)₂O), for example, about 1.1equivalents, under a hydrogen atmosphere in the presence of a transitionmetal catalyst to provide the Boc protected intermediate 20,3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester. Thereaction is typically conducted at ambient temperature for about 12 toabout 72 hours. Finally, intermediate 20 is contacted with a largeexcess, for example at least about 25 equivalents, of ammonium formatein an inert diluent, such as methanol, in the presence of a transitionmetal catalyst to provide the product 16 in the endo configuration, withhigh stereospecificity, for example endo to exo ratio of >99:1. Thereaction is typically conducted at ambient temperature for about 12 toabout 72 hours or until the reaction is substantially complete. It isadvantageous to add the ammonium formate reagent in portions. Forexample, intermediate 20 is contacted with an initial portion ofammonium formate of about 15 to about 25 equivalents. After an intervalof about 12 to about 36 hours, an additional portion of about 5 to about10 equivalents of ammonium formate is added. The subsequent addition canbe repeated after a similar interval. The product 16 can be purified byconventional procedures, such as alkaline extraction.

The quinolinone carboxylic acid 14 is readily prepared by proceduressimilar to those reported in the literature in Suzuki et al,Heterocycles, 2000, 53, 2471-2485 and described in the examples below.

The oxirane intermediate 12 used in Scheme C can be prepared by reactionwith a halomethyloxirane as shown in Scheme G:

where L is a halo leaving group. This reaction is typically conducted bycontacting the amine of formula 2 with between about 1 and about 2equivalents of a halomethyloxirane in a polar diluent such as ethanol.The reaction is typically conducted at ambient temperature for betweenabout 12 and about 24 hours, or until the reaction is substantiallycomplete. The linear intermediate 21 is typically isolated byconventional procedures as a solid. The solid 21 is typically dissolvedin an inert diluent, for example tetrahydrofuran, in the presence of amolar excess of base, for example sodium hydroxide, to produce thecyclized form 12.

The secondary amines H—Y are available commercially or are readilysynthesized from common starting materials according to standardprotocols described in the literature or in textbooks, such as J. March,Advanced Organic Chemistry, Fourth Edition, Wiley, New York, 1992, andas exemplified below.

In yet another alternative method of synthesis, compounds of formula (D)are prepared by coupling the substituted quinolinone carboxylic acid 14with an intermediate of formula 22 as illustrated in Scheme H.

The reaction of Scheme H is typically conducted under the amide couplingconditions described above for the reaction of the carboxylic acid 14with intermediate 15.

Intermediates of formula 22 can be prepared by deprotecting anintermediate of formula 23:

where P² represents an amino-protecting group.

Intermediates of formula 23 can be prepared from readily availablestarting materials using procedures analogous to the reactions describedabove and/or using alternative reactions well known to those skilled inthe art. For example, intermediate 23 can be prepared using anintermediate 24

which may be formed by protecting the amino nitrogen of theaminoazobicyclooctane 15 with amino-protecting group P² and thenremoving P¹ from the nitrogen of the azabicyclooctane group. Protectinggroups P¹ and P² are chosen such that they are removed under differentconditions. For example when P¹ is chosen as Boc, then Cbz can be usedas P². Substituting the protected aminotropane 24 for intermediate 1 inthe reactions described in Schemes A, C, and D provides intermediates offormula 23.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereto are described in the examples below.

Accordingly, in a method aspect, the invention provides a process forpreparing a compound of formula (I), wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷,n, and X are defined as in formula (I), or a salt or stereoisomerthereof, the process comprising:

(a) reacting a compound of formula (VI):

wherein L′ is an anion, with a compound of formula (VII):

or

(b) reacting a compound of formula (VIII):

with a compound of formula (IX):

to provide a compound of formula (I), or a salt or stereoisomer thereof.

The invention also provides a process for preparing a compound offormula (I), wherein R³ is hydroxy and R¹, R², R⁴, R⁵, R⁶, R⁷, n, and Xare defined as in formula (I), or a salt or stereoisomer thereof, theprocess comprising:

step (a) or step (b) as defined above, or

(c) reacting a compound of formula (X):

or a salt thereof, with a compound of formula (VII) and a compound offormula (XI):

wherein L is a leaving group;or

(d) reacting a compound of formula (X) with a compound of formula (XII):

to provide a compound of formula (I), or a salt or stereoisomer thereof.

In still other aspects, this invention is directed to additionalprocesses described herein; and to the products prepared by any of theprocesses described herein.

Pharmaceutical Compositions

The quinolinone-carboxamide compounds of the invention are typicallyadministered to a patient in the form of a pharmaceutical composition.Such pharmaceutical compositions may be administered to the patient byany acceptable route of administration including, but not limited to,oral, rectal, vaginal, nasal, inhaled, topical (including transdermal)and parenteral modes of administration.

Accordingly, in one of its compositions aspects, the invention isdirected to a pharmaceutical composition comprising apharmaceutically-acceptable carrier or excipient and a therapeuticallyeffective amount of a compound of formula (I) or a pharmaceuticallyacceptable salt thereof. Optionally, such pharmaceutical compositionsmay contain other therapeutic and/or formulating agents if desired.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the present inventionor a pharmaceutically-acceptable salt thereof. Typically, suchpharmaceutical compositions will contain from about 0.1 to about 95% byweight of the active agent; preferably, from about 5 to about 70% byweight; and more preferably from about 10 to about 60% by weight of theactive agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the ingredients for such compositionsare commercially-available from, for example, Sigma, P.O. Box 14508, St.Louis, Mo. 63178. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, such as microcrystallinecellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical compositions.

The pharmaceutical compositions of the invention are typically preparedby thoroughly and intimately mixing or blending a compound of theinvention with a pharmaceutically-acceptable carrier and one or moreoptional ingredients. If necessary or desired, the resulting uniformlyblended mixture can then be shaped or loaded into tablets, capsules,pills and the like using conventional procedures and equipment.

The pharmaceutical compositions of the invention are preferably packagedin a unit dosage form. The term “unit dosage form” refers to aphysically discrete unit suitable for dosing a patient, i.e., each unitcontaining a predetermined quantity of active agent calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be capsules, tablets, pills, and the like.

In a preferred embodiment, the pharmaceutical compositions of theinvention are suitable for oral administration. Suitable pharmaceuticalcompositions for oral administration may be in the form of capsules,tablets, pills, lozenges, cachets, dragees, powders, granules; or as asolution or a suspension in an aqueous or non-aqueous liquid; or as anoil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup;and the like; each containing a predetermined amount of a compound ofthe present invention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof the invention will typically comprise a compound of the presentinvention as the active ingredient and one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate. Optionally or alternatively, such solid dosageforms may also comprise: (1) fillers or extenders, such as starches,microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants,such as glycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and/or sodium carbonate; (5) solution retarding agents, such asparaffin; (6) absorption accelerators, such as quaternary ammoniumcompounds; (7) wetting agents, such as cetyl alcohol and/or glycerolmonostearate; (8) absorbents, such as kaolin and/or bentonite clay; (9)lubricants, such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and/or mixtures thereof;(10) coloring agents; and (11) buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically-acceptable antioxidants include: (1) water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal-chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like. Coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate (CAP), polyvinyl acetatephthalate (PVAP), hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate(CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical compositions of the present invention mayalso be formulated to provide slow or controlled release of the activeingredient using, by way of example, hydroxypropyl methyl cellulose invarying proportions; or other polymer matrices, liposomes and/ormicrospheres.

In addition, the pharmaceutical compositions of the present inventionmay optionally contain opacifying agents and may be formulated so thatthey release the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Such liquid dosage formstypically comprise the active ingredient and an inert diluent, such as,for example, water or other solvents, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (esp., cottonseed, groundnut, corn, germ,olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Alternatively, the pharmaceutical compositions of the invention areformulated for administration by inhalation. Suitable pharmaceuticalcompositions for administration by inhalation will typically be in theform of an aerosol or a powder. Such compositions are generallyadministered using well-known delivery devices, such as a metered-doseinhaler, a dry powder inhaler, a nebulizer or a similar delivery device.

When administered by inhalation using a pressurized container, thepharmaceutical compositions of the invention will typically comprise theactive ingredient and a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.

Additionally, the pharmaceutical composition may be in the form of acapsule or cartridge (made, for example, from gelatin) comprising acompound of the invention and a powder suitable for use in a powderinhaler. Suitable powder bases include, by way of example, lactose orstarch.

The compounds of the invention can also be administered transdermallyusing known transdermal delivery systems and excipients. For example, acompound of the invention can be admixed with permeation enhancers, suchas propylene glycol, polyethylene glycol monolaurate,azacycloalkan-2-ones and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention:

FORMULATION EXAMPLE A

Hard gelatin capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 50 mg Lactose (spray-dried)200 mg Magnesium stearate 10 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a hard gelatin capsule (260 mg of        composition per capsule).

FORMULATION EXAMPLE B

Hard gelatin capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 20 mg Starch 89 mgMicrocrystalline cellulose 89 mg Magnesium stearate 2 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then passed through a No. 45 mesh U.S. sieve and loaded into        a hard gelatin capsule (200 mg of composition per capsule).

FORMULATION EXAMPLE C

Capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 10 mg Polyoxyethylenesorbitan monooleate 50 mg Starch powder 250 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a gelatin capsule (310 mg of composition        per capsule).

FORMULATION EXAMPLE D

Tablets for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 5 mg Starch 50 mgMacrocrystalline cellulose 35 mg Polyvinylpyrrolidone (10 wt. % inwater) 4 mg Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mgTalc 1 mg

-   -   Representative Procedure: The active ingredient, starch and        cellulose are passed through a No. 45 mesh U.S. sieve and mixed        thoroughly. The solution of polyvinylpyrrolidone is mixed with        the resulting powders, and this mixture is then passed through a        No. 14 mesh U.S. sieve. The granules so produced are dried at        50-60° C. and passed through a No. 18 mesh U.S. sieve. The        sodium carboxymethyl starch, magnesium stearate and talc        (previously passed through a No. 60 mesh U.S. sieve) are then        added to the granules. After mixing, the mixture is compressed        on a tablet machine to afford a tablet weighing 100 mg.

FORMULATION EXAMPLE E

Tablets for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 25 mg Microcrystallinecellulose 400 mg Silicon dioxide fumed 10 mg Stearic acid 5 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then compressed to form tablets (440 mg of composition per        tablet).

FORMULATION EXAMPLE F

Single-scored tablets for oral administration are prepared as follows:

Ingredients Amount Compound of the invention 15 mg Cornstarch 50 mgCroscarmellose sodium 25 mg Lactose 120 mg Magnesium stearate 5 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and compressed to form a single-scored tablet (215 mg of        compositions per tablet).

FORMULATION EXAMPLE G

A suspension for oral administration is prepared as follows:

Ingredients Amount Compound of the invention 0.1 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum k(Vanderbilt Co.) 1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilledwater q.s. to 100 mL

-   -   Representative Procedure: The ingredients are mixed to form a        suspension containing 10 mg of active ingredient per 10 mL of        suspension.

FORMULATION EXAMPLE H

A dry powder for administration by inhalation is prepared as follows:

Ingredients Amount Compound of the invention 1.0 mg Lactose 25 mg

-   -   Representative Procedure: The active ingredient is micronized        and then blended with lactose. This blended mixture is then        loaded into a gelatin inhalation cartridge. The contents of the        cartridge are administered using a powder inhaler.

FORMULATION EXAMPLE I

A dry powder for administration by inhalation in a metered dose inhaleris prepared as follows:

-   -   Representative Procedure: A suspension containing 5 wt. % of a        compound of the invention and 0.1 wt. % lecithin is prepared by        dispersing 10 g of active compound as micronized particles with        mean size less than 10 μm in a solution formed from 0.2 g of        lecithin dissolved in 200 mL of demineralized water. The        suspension is spray dried and the resulting material is        micronized to particles having a mean diameter less than 1.5 μm.        The particles are loaded into cartridges with pressurized        1,1,1,2-tetrafluoroethane.

FORMULATION EXAMPLE J

An injectable formulation is prepared as follows:

Ingredients Amount Compound of the invention 0.2 g Sodium acetate buffersolution (0.4 M) 40 mL HCl (0.5 N) or NaOH (0.5 N) q.s. to pH 4 Water(distilled, sterile) q.s. to 20 mL

-   -   Representative Procedure: The above ingredients are blended and        the pH is adjusted to 4±0.5 using 0.5 N HCl or 0.5 N NaOH.

FORMULATION EXAMPLE K

Capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the Invention 4.05 mg Microcrystallinecellulose (Avicel PH 103) 259.2 mg Magnesium stearate 0.75 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a gelatin capsule (Size #1, White, Opaque)        (264 mg of composition per capsule).

FORMULATION EXAMPLE L

Capsules for oral administration are prepared as follows:

Ingredients Amount Compound of the Invention 8.2 mg Microcrystallinecellulose (Avicel PH 103) 139.05 mg Magnesium stearate 0.75 mg

-   -   Representative Procedure: The ingredients are thoroughly blended        and then loaded into a gelatin capsule (Size #1, White, Opaque)        (148 mg of composition per capsule).

It will be understood that any form of the compounds of the invention,(i.e. free base, pharmaceutical salt, or solvate) that is suitable forthe particular mode of administration, can be used in the pharmaceuticalcompositions discussed above.

Utility

The quinolinone-carboxamide compounds of the invention are 5-HT₄receptor agonists and therefore are expected to be useful for treatingmedical conditions mediated by 5-HT₄ receptors or associated with 5-HT₄receptor activity, i.e. medical conditions which are ameliorated bytreatment with a 5-HT₄ receptor agonist. Such medical conditionsinclude, but are not limited to, irritable bowel syndrome (IBS), chronicconstipation, functional dyspepsia, delayed gastric emptying,gastroesophageal reflux disease (GERD), gastroparesis, diabetic andidiopathic gastropathy, post-operative ileus, intestinalpseudo-obstruction, and drug-induced delayed transit. In addition, ithas been suggested that some 5-HT₄ receptor agonist compounds may beused in the treatment of central nervous system disorders includingcognitive disorders, behavioral disorders, mood disorders, and disordersof control of autonomic function.

In particular, the compounds of the invention increase motility of thegastrointestinal (GI) tract and thus are expected to be useful fortreating disorders of the GI tract caused by reduced motility inmammals, including humans. Such GI motility disorders include, by way ofillustration, chronic constipation, constipation-predominant irritablebowel syndrome (C-IBS), diabetic and idiopathic gastroparesis, andfunctional dyspepsia.

In one aspect, therefore, the invention provides a method of increasingmotility of the gastrointestinal tract in a mammal, the methodcomprising administering to the mammal a therapeutically effectiveamount of a pharmaceutical composition comprising apharmaceutically-acceptable carrier and a compound of the invention.

When used to treat disorders of reduced motility of the GI tract orother conditions mediated by 5-HT₄ receptors, the compounds of theinvention will typically be administered orally in a single daily doseor in multiple doses per day, although other forms of administration maybe used. The amount of active agent administered per dose or the totalamount administered per day will typically be determined by a physician,in the light of the relevant circumstances, including the condition tobe treated, the chosen route of administration, the actual compoundadministered and its relative activity, the age, weight, and response ofthe individual patient, the severity of the patient's symptoms, and thelike.

Suitable doses for treating disorders of reduced motility of the GItract or other disorders mediated by 5-HT₄ receptors will range fromabout 0.0007 to about 20 mg/kg/day of active agent, preferably fromabout 0.0007 to about 1 mg/kg/day. For an average 70 kg human, thiswould amount to from about 0.05 to about 70 mg per day of active agent.

In one aspect of the invention, the compounds of the invention are usedto treat chronic constipation. When used to treat chronic constipation,the compounds of the invention will typically be administered orally ina single daily dose or in multiple doses per day. Preferably, the dosefor treating chronic constipation will range from about 0.05 to about 70mg per day.

In another aspect of the invention, the compounds of the invention areused to treat irritable bowel syndrome. When used to treatconstipation-predominant irritable bowel syndrome, the compounds of theinvention will typically be administered orally in a single daily doseor in multiple doses per day. Preferably, the dose for treatingconstipation-predominant irritable bowel syndrome will range from about0.05 to about 70 mg per day.

In another aspect of the invention, the compounds of the invention areused to treat diabetic gastroparesis. When used to treat diabeticgastroparesis, the compounds of the invention will typically beadministered orally in a single daily dose or in multiple doses per day.Preferably, the dose for treating diabetic gastroparesis will range fromabout 0.05 to about 70 mg per day.

In yet another aspect of the invention, the compounds of the inventionare used to treat functional dyspepsia. When used to treat functionaldyspepsia, the compounds of the invention will typically be administeredorally in a single daily dose or in multiple doses per day. Preferably,the dose for treating functional dyspepsia will range from about 0.05 toabout 70 mg per day.

The invention also provides a method of treating a mammal having adisease or condition associated with 5-HT₄ receptor activity, the methodcomprising administering to the mammal a therapeutically effectiveamount of a compound of the invention or of a pharmaceutical compositioncomprising a compound of the invention.

As described above, compounds of the invention are 5-HT₄ receptoragonists. The invention further provides, therefore, a method ofagonizing a 5-HT₄ receptor in a mammal, the method comprisingadministering a compound of the invention to the mammal. In addition,the compounds of the invention are also useful as research tools forinvestigating or studying biological systems or samples having 5-HT₄receptors, or for discovering new 5-HT₄ receptor agonists. Moreover,since compounds of the invention exhibit binding selectivity for 5-HT₄receptors as compared with binding to receptors of other 5-HT subtypes,particularly 5-HT₃ receptors, such compounds are particularly useful forstudying the effects of selective agonism of 5-HT₄ receptors in abiological system or sample. Any suitable biological system or samplehaving 5-HT₄ receptors may be employed in such studies which may beconducted either in vitro or in vivo. Representative biological systemsor samples suitable for such studies include, but are not limited to,cells, cellular extracts, plasma membranes, tissue samples, mammals(such as mice, rats, guinea pigs, rabbits, dogs, pigs, etc.) and thelike.

In this aspect of the invention, a biological system or samplecomprising a 5-HT₄ receptor is contacted with a 5-HT₄ receptor-agonizingamount of a compound of the invention. The effects of agonizing the5-HT₄ receptor are then determined using conventional procedures andequipment, such as radioligand binding assays and functional assays.Such functional assays include ligand-mediated changes in intracellularcyclic adenosine monophosphate (cAMP), ligand-mediated changes inactivity of the enzyme adenylyl cyclase (which synthesizes cAMP),ligand-mediated changes in incorporation of analogs of guanosinetriphosphate (GTP), such as [³⁵S]GTPγS (guanosine5′-O-(γ-thio)triphosphate) or GTP-Eu, into isolated membranes viareceptor catalyzed exchange of GTP analogs for GDP analogs,ligand-mediated changes in free intracellular calcium ions (measured,for example, with a fluorescence-linked imaging plate reader or FLIPR®from Molecular Devices, Inc.), and measurement of mitogen activatedprotein kinase (MAPK) activation. A compound of the invention mayagonize or increase the activation of 5-HT₄ receptors in any of thefunctional assays listed above, or assays of a similar nature. A 5-HT₄receptor-agonizing amount of a compound of the invention will typicallyrange from about 1 nanomolar to about 1000 nanomolar.

Additionally, the compounds of the invention can be used as researchtools for discovering new 5-HT₄ receptor agonists. In this embodiment,5-HT₄ receptor binding or functional data for a test compound or a groupof test compounds is compared to the 5-HT₄ receptor binding orfunctional data for a compound of the invention to identify testcompounds that have superior binding or functional activity, if any.This aspect of the invention includes, as separate embodiments, both thegeneration of comparison data (using the appropriate assays) and theanalysis of the test data to identify test compounds of interest.

Among other properties, compounds of the invention have been found to bepotent agonists of the 5-HT₄ receptor and to exhibit substantialselectivity for the 5-HT₄ receptor subtype over the 5-HT₃ receptorsubtype in radioligand binding assays. Further, compounds of theinvention of which particular mention was made have demonstratedsuperior pharmacokinetic properties in a rat model. Such compounds arethus expected to be highly bioavailable upon oral administration. Inaddition, these compounds have been shown not to exhibit an unacceptablelevel of inhibition of the potassium ion current in an in vitrovoltage-clamp model using isolated whole cells expressing the hERGcardiac potassium channel. The voltage-clamp assay is an acceptedpre-clinical method of assessing the potential for pharmaceutical agentsto change the pattern of cardiac repolarization, specifically to cause,so-called QT prolongation, which has been associated with cardiacarrhythmia. (Cavero et al., Opinion on Pharmacotherapy, 2000, 1, 947-73,Fermini et al., Nature Reviews Drug Discovery, 2003, 2, 439-447)Accordingly, pharmaceutical compositions comprising these compounds ofthe invention are expected to have an acceptable cardiac profile.

There properties, as well as the utility of the compounds of theinvention, can be demonstrated using various in vitro and in vivo assayswell-known to those skilled in the art. Representative assays aredescribed in further detail in the following examples.

EXAMPLES

The following synthetic and biological examples are offered toillustrate the invention, and are not to be construed in any way aslimiting the scope of the invention. In the examples below, thefollowing abbreviations have the following meanings unless otherwiseindicated. Abbreviations not defined below have their generally acceptedmeanings.

-   -   Boc=tert-butoxycarbonyl    -   (Boc)₂O=di-tert-butyl dicarbonate    -   DCM=dichloromethane    -   DMF=N,N-dimethylformamide    -   DMSO=dimethyl sulfoxide    -   EtOAc=ethyl acetate    -   mCPBA=m-chloroperbenzoic acid    -   MeCN=acetonitrile    -   MTBE=tert-butyl methyl ether    -   PyBop=benzotriazol-1-yloxytripyrrolidino-phosphonium        hexafluorophosphate    -   R_(f)=retention factor    -   RT=room temperature    -   TFA=trifluoroacetic acid    -   THF=tetrahydrofuran

Reagents (including secondary amines) and solvents were purchased fromcommercial suppliers (Aldrich, Fluka, Sigma, etc.), and used withoutfurther purification. Reactions were run under nitrogen atmosphere,unless noted otherwise. Progress of reaction mixtures was monitored bythin layer chromatography (TLC), analytical high performance liquidchromatography (anal. HPLC), and mass spectrometry, the details of whichare given below and separately in specific examples of reactions.Reaction mixtures were worked up as described specifically in eachreaction; commonly they were purified by extraction and otherpurification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by preparative HPLC: a general protocol is describedbelow. Characterization of reaction products was routinely carried outby mass and ¹H-NMR spectrometry. For NMR measurement, samples weredissolved in deuterated solvent (CD₃OD, CDCl₃, or DMSO-d₆), and ¹H-NMRspectra were acquired with a Varian Gemini 2000 instrument (300 MHz)under standard observation conditions. Mass spectrometric identificationof compounds was performed by an electrospray ionization method (ESMS)with a Perkin Elmer instrument (PE SCIEX API 150 EX).

General Protocol for Analytical HPLC

Crude compounds were dissolved in 50% MeCN/H₂O (with 0.1% TFA) at0.5-1.0 mg/mL concentration, and analyzed using the followingconditions:

Column: Zorbax Bonus-RP (3.5 μm of particle size, 2.1 × 50 mm) Flowrate: 0.5 mL/min Mobile Phases: A = 90% MeCN/10% H₂O/0.1% TFA B = 98%H₂O/2% MeCN/0.1% TFA Gradient: 10% A/90% B (0-0.5 min); 10% A/90% B to50% A/50% B (linear, 0.5-5 min) Detector wavelength: 214, 254, and 280nm.Alternative conditions, when used, are indicated explicitly.General Protocol for Preparative HPLC Purification

Crude compounds were dissolved in 50% acetic acid in water at 50-100mg/mL concentration, filtered, and fractionated using the followingprocedure:

Column: YMC Pack-Pro C18 (50a × 20 mm; ID = 5 μm) Flow rate: 40 mL/minMobile Phases: A = 90% MeCN/10% H₂O/0.1% TFA B = 98% H₂O/2% MeCN/0.1%TFA Gradient: 10% A/90% B to 50% A/50% B over 30 min (linear) Detectorwavelength: 214 nm.Preparation of Secondary Amines

Preparation of Secondary Amines not Available Commercially isExemplified by the following:

Thiomorpholine-1,1-dioxide was prepared from thiomorpholine byprotection of the secondary amine to N-Boc thiomorpholine ((Boc)₂O,MeOH), oxidation to sulfone (mCPBA, CH₂Cl₂, 0° C.), and deprotection ofthe N-Boc group to provide the free amine (CF₃CO₂H, CH₂Cl₂). (m/z):[M+H]⁺ calcd for C₄H₉NO₂S, 136.04; found, 135.9.

The N-sulfonyl derivatives of piperazine were prepared from N-Bocpiperazine by reacting with respective sulfonyl chloride (iPr₂NEt,CH₂Cl₂, 0° C.), and deprotecting the N-Boc group (CF₃CO₂H, CH₂Cl₂).1-Methanesulfonyl-piperazine: ¹H-NMR (CDCl₃; neutral): δ (ppm) 3.1 (t,4H), 2.9 (t, 4H), 2.7 (s, 3H).1-(Methylsulfonyl)methanesulfonyl-piperazine: ¹H-NMR (CD₃OD): δ (ppm)2.90 (s, 3H), 3.02 (m, 4H), 3.38 (m, 4H), 4.61 (s, 2H).Methanesulfonylpiperazine was also prepared by reacting methanesulfonylchloride with excess piperazine (>2 equivalents) in water.

The racemic or single chiral isomer forms of 3-acetylaminopyrrolidinewere prepared by treating N¹-Boc-3-aminopyrrolidine (racemate, 3R, or3S) with acetyl chloride (iPr₂NEt, CH₂Cl₂, 0° C.), and deprotecting theN-Boc group (CF₃CO₂H, CH₂Cl₂). 3-(Acetamido)pyrrolidine: ¹H-NMR(DMSO-d₆; TFA salt): δ (ppm) 4.2 (quin, 1H), 3.3-3.1 (m, 3H), 2.9 (m,1H), 2.0 (m, 1H), 1.8 (br s, 4H).

3-((R)-2-Hydroxypropionamido)pyrrolidine was prepared after amidation ofN¹-Boc-3-aminopyrrolidine (L-lactic acid, PyBOP, DMF, RT), anddeprotection of N-Boc group (CF₃CO₂H, CH₂Cl₂). (m/z): [M+H]⁺ calcd forC₇H₁₄N₂O₂, 159.11; found, 159.0. ¹H-NMR (CD₃OD; TFA salt): δ (ppm) 4.4(quin, 1H), 4.1 (q, 1H), 3.5-3.4 (m, 2H), 3.3-3.2 (m, 2H), 2.3 (m, 1H),2.0 (m, 1H), 1.3 (d, 3H).

The N³-alkanesulfonyl derivatives of (3R)-aminopyrrolidine were obtainedby treating N¹-Boc-(3R)-aminopyrrolidine with propionylsulfonyl chlorideor cyclohexylmethylsulfonyl chloride (i-Pr₂NEt, CH₂Cl₂, 0° C.), anddeprotecting N-Boc group (CF₃CO₂H, CH₂Cl₂).

3-(N-Acetyl-N-methylamido)piperidine was prepared from N³-Cbz protected3-amino-piperidine-1-carboxylic acid t-butyl ester (De Costa, B., et al.J. Med. Chem. 1992, 35, 4334-43) after four synthetic steps: i) MeI,n-BuLi, THF, −78° C. to rt; ii) H₂ (1 atm), 10% Pd/C, EtOH; iii) AcCl,i-Pr₂NEt, CH₂Cl₂; iv) CF₃CO₂H, CH₂Cl₂. m/z: [M+H]⁺ calcd for C₈H₁₆N₂O:157.13; found, 157.2. ¹H-NMR (CD₃OD; TFA salt): δ (ppm) 4.6 (m, 1H), 3.3(m, 1H), 3.2 (m, 1H), 3.0 (m, 1H), 2.9 (s, 3H), 2.8 (m, 1H), 2.0 (s,3H), 1.9-1.7 (m, 4H).

3-(N-Acetyl-amido)piperidine was prepared from3-amino-piperidine-1-carboxylic acid tert-butyl ester afterN-acetylation and deprotection of the N-Boc group: i) AcCl, i-Pr₂NEt,CH₂Cl₂; ii) CF₃CO₂H, CH₂Cl₂. ¹H-NMR (CD₃OD; TFA salt): δ (ppm) 3.9 (m,1H), 3.3 (dd, 1H), 3.2 (m, 1H), 2.9 (dt, 1H), 2.75 (dt, 1H), 2.0-1.9 (m,2H), 1.9 (s, 3H), 1.8-1.4 (m, 2H).

The N³-alkanesulfonyl derivatives of 3-aminopiperidine were synthesizedby reacting the chiral or racemic forms of3-amino-piperidine-1-carboxylic acid tert-butyl ester with therespective alkanesulfonyl chloride (i-Pr₂NEt, CH₂Cl₂) and deprotectingthe N-Boc group (CF₃CO₂H, CH₂Cl₂).(3S)-3-(ethanesulfonylamido)piperidine: ¹H-NMR (CD₃OD): δ (ppm) 1.29(t,3H, J₁=7.4 Hz), 1.50-1.80 (m, 2H), 1.90-2.10 (m, 2H), 2.89 (m, 2H), 3.05(q, 2H, J₁=7.4 Hz), 3.27 (m, 2H), 3.40 (d of d(br), 1H), 3.52 (m, 1H).3S-Methylsulfonylmethanesulfonylamido-piperidine: ¹H-NMR (CD₃OD): δ(ppm) 2.13-2.30 (m, 2H), 2.40-2.57 (m, 2H), 2.98 (m, 2H), 3.15 (s, 3H),3.21 (m, 2H), 3.30 (br d, 1H), 3.74 (m, 1H).

3-(Methylamino)-1-acetylpyrrolidine was prepared from3-(methylamino)-1-benzylpyrrolidine (TCI America) after four steps: i)(Boc)₂O, MeOH, rt; ii) H₂ (1 atm), 10% Pd/C, EtOH; iii) AcCl, i-Pr₂NEt,CH₂Cl₂; iv) CF₃CO₂H, CH₂Cl₂. (m/z): [M+H]⁺ calcd for C₇H₁₄N₂O: 143.12;found, 143.0.

3-(Methylamino)-1-(methanesulfonyl)pyrrolidine was prepared from3-(methylamino)-1-benzylpyrrolidine after four steps: i) (Boc)₂O, MeOH,rt; ii) H₂ (1 atm), 10% Pd/C, EtOH; iii) CH₃SO₂Cl, i-Pr₂NEt, CH₂Cl₂; iv)CF₃CO₂H, CH₂Cl₂. (m/z): [M+H]⁺ calcd for C₆H₁₄N₂O₂S: 179.08; found,179.2. 3R-Methylamino-1-(methanesulfonyl)pyrrolidine was prepared in asimilar manner from (3R)-(methylamino)-1-benzylpyrrolidine.

Derivatives of tetrahydro-3-thiophenamine-1,1-dioxide were preparedfollowing the protocol of Loev, B. J. Org. Chem. 1961, 26, 4394-9 byreacting 3-sulfolene with a requisite primary amine in methanol (cat.KOH, rt). N-Methyl-3-tetrahydrothiopheneamine-1,1-dioxide (TFA salt):¹H-NMR (DMSO-d₆): δ (ppm) 9.4 (br s, 2H), 4.0-3.8 (quin, 1H), 3.6-3.5(dd, 1H), 3.4-3.3 (m, 1H), 3.2-3.1 (m, 2H), 2.5 (s, 3H), 2.4 (m, 1H),2.1 (m, 1H).N-2-(1-hydroxy)ethyl-3-tetrahydrothiopheneamine-1,1-dioxide: (m/z):[M+H]⁺ calcd for C₆H₁₃NO₃S: 180.07; found, 180.2.

N-Methyl-tetrahydro-2H-thiopyran-4-amine-1,1-dioxide was prepared fromtetrahydro-4H-thiopyran-4-one: i) MeNH₂, NaBH₄; ii) (Boc)₂O, MeOH; iii)mCPBA, CH₂Cl₂, 0° C.; iv) CF₃CO₂H, CH₂Cl₂. (m/z): [M+H]⁺ calcd forC₆H₁₃NO₂S 164.07; found, 164.9. ¹H-NMR (CD₃OD; TFA salt): δ (ppm)3.4-3.1 (m, 5H), 2.7 (s, 3H), 2.4 (br d, 2H), 2.1 (br m, 2H).

1-Acetyl-3-(methylamino)piperidine was prepared from N³-Cbz protected3-methylamino-piperidine: i) AcCl, i-Pr₂NEt, CH₂Cl₂; ii) H₂ (1 atm), 10%Pd/C, EtOH. 1H-NMR (CD₃OD): δ (ppm) 4.0 (m, 1H), 3.6 (m, 1H), 3.4-3.2(m, 2H), 3.0 (m, 1H), 2.6 (s, 3H), 2.1 (s, 3H), 1.8-1.6 (m, 4H).

1-(Methanesulfonyl)-3-(methylamino)piperidine was prepared from N³-Cbzprotected 3-methylamino-piperidine: i) CH₃SO₂Cl, i-Pr₂NEt, CH₂Cl₂; ii)H₂ (1 atm), 10% Pd/C, EtOH. (m/z): [M+H]⁺ calcd for C₇H₁₆N₂O₂S 193.10;found, 193.0. ¹H-NMR (DMSO-d₆; TFA salt): δ (ppm) 3.4 (dd, 1H), 3.2 (m,2H), 3.10 (s, 3H), 3.0-2.9 (m, 2H), 2.8 (s, 3H), 1.85-1.75 (m, 2H),1.6-1.4 (m, 2H).

The N-derivatives of piperazine such as 1-(methoxycarbonyl)piperazine,1-(dimethylaminocarbonyl)piperazine, and1-(dimethylaminosulfonyl)piperazine were prepared by reacting piperazinewith methylchloroformate, dimethylaminochoroformate, ordimethylaminosulfamoyl chloride, respectively.

1-Methylamino-2-methylsulfonylethane was obtained by reactingmethylamine with methyl vinyl sulfone in methanol.

N-[2-(2-methoxyethylamino)ethyl], N-methyl-methanesulfonamide wassynthesized starting from partially N-Boc protected ethanediamine by thefollowing four step reaction sequence: i) methylsulfonyl chloride,triethylamine; ii) MeI, Cs₂CO₃; iii) NaH, 1-bromo-2-methoxyethane; iv)CF₃CO₂H.

Methyl 4-piperidinylcarbamate was prepared from the reaction of N¹-Bocprotected 4-aminopiperidine with methylchloroformate followed by thedeprotection of the N-Boc group.

4-Piperidinol-dimethylcarbamate, and N-dimethyl-N′-(3-piperidinyl)ureawere prepared by reacting dimethylcarbamoyl chloride with N-Bocprotected 4-piperidinol or N¹-Boc-3-aminopiperidine, respectively.

3-(Methylamino)-1-(dimethylaminosulfonyl)pyrrolidine was obtained byreacting 3-(N-methyl-N-Boc-amino)pyrrolidine with dimethylsulfamoylchloride.

2-(3-Pyrrolidinyl)isothiazolidine-1,1-dioxide was synthesized bytreating N¹-Boc protected 3-aminopyrrolidine with 3-chloropropylsulfonylchloride in the presence of triethylamine, and followed by deprotectionof the Boc group by treatment with trifluoroacetic acid.

Preparation 1 (1S,3R,5R)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylicacid tert-butyl ester

a. Preparation of 8-benzyl-8-azabicyclo[3.2.1]octan-3-one

Concentrated hydrochloric acid (30 mL) was added to a heterogeneoussolution of 2,5-dimethoxy tetrahydrofuran (82.2 g, 0.622 mol) in water(170 mL) while stirring. In a separate flask cooled to 0° C. (ice bath),concentrated hydrochloric acid (92 mL) was added slowly to a solution ofbenzyl amine (100 g, 0.933 mol) in water (350 mL). The2,5-dimethoxytetrahydrofuran solution was stirred for approximately 20min, diluted with water (250 mL), and then the benzyl amine solution wasadded, followed by the addition of a solution of 1,3-acetonedicarboxylicacid (100 g, 0.684 mol) in water (400 mL) and then the addition ofsodium hydrogen phosphate (44 g, 0.31 mol) in water (200 mL). The pH wasadjusted from pH 1 to pH ˜4.5 using 40% NaOH. The resulting cloudy andpale yellow solution was stirred overnight. The solution was thenacidified to pH 3 from pH 7.5 using 50% hydrochloric acid, heated to 85°C. and stirred for 2 hours. The solution was cooled to room temperature,basified to pH 12 using 40% NaOH, and extracted with DCM (3×500 mL). Thecombined organic layers were washed with brine, dried (MgSO₄), filteredand concentrated under reduced pressure to produce the crude titleintermediate as a viscous brown oil (52 g).

To a solution of the crude intermediate in methanol (1000 mL) was addeddi-tert-butyl dicarbonate (74.6 g, 0.342 mol) at 0° C. The solution wasallowed to warm to room temperature and stirred overnight. The methanolwas removed under reduced pressure and the resulting oil was dissolvedin dichloromethane (1000 mL). The intermediate was extracted into 1 MH₃PO₄ (1000 mL) and washed with dichloromethane (3×250 mL) The aqueouslayer was basified to pH 12 using aqueous NaOH, and extracted withdichloromethane (3×500 mL). The combined organic layers were dried(MgSO₄), filtered and concentrated under reduced pressure to produce thetitle intermediate as a viscous, light brown oil. ¹H-NMR (CDCl₃) δ (ppm)7.5-7.2 (m, 5H, C₆H₅), 3.7 (s, 2H, CH₂Ph), 3.45 (broad s, 2H, CH—NBn),2.7-2.6 (dd, 2H, CH₂CO), 2.2-2.1 (dd, 2H, CH₂CO), 2.1-2.0 (m, 2H,CH₂CH₂), 1.6 (m, 2H, CH₂CH₂). (m/z): [M+H]⁺ calcd for C₁₄H₁₇NO 216.14;found, 216.0.

b. Preparation of 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylic acidtert-butyl ester

To a solution of 8-benzyl-8-azabicyclo[3.2.1]octan-3-one (75 g, 0.348mol) in EtOAc (300 mL) was added a solution of di-tert-butyl dicarbonate(83.6 g, 0.383 mol, 1.1 eq) in EtOAc (300 mL). The resulting solutionand rinse (100 mL EtOAc) was added to a 1 L Parr hydrogenation vesselcontaining 23 g of palladium hydroxide (20 wt. % Pd, dry basis, oncarbon, ˜50% wet with water; e.g. Pearlman's catalyst) under a stream ofnitrogen. The reaction vessel was degassed (alternating vacuum and N₂five times) and pressurized to 60 psi of H₂ gas. The reaction solutionwas agitated for two days and recharged with H₂ as needed to keep the H₂pressure at 60 psi until the reaction was complete as monitored bysilica thin layer chromatography. The black solution was then filteredthrough a pad of Celite® and concentrated under reduced pressure toyield the title intermediate quantitatively as a viscous, yellow toorange oil. It was used in the next step without further treatment. ¹HNMR (CDCl₃)

(ppm) 4.5 (broad, 2H, CH—NBoc), 2.7 (broad, 2H, CH₂CO), 2.4-2.3 (dd, 2H,CH₂C₁H₂), 2.1 (broad m, 2H, CH₂CO), 1.7-1.6 (dd, 2H, CH₂CH₂), 1.5 (s,9H, (CH₃)₃COCON)).

c. Preparation of(1S,3R,5R)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylic acidtert-butyl ester

To a solution of the product of the previous step (75.4 g, 0.335 mol) inmethanol (1 L) was added ammonium formate (422.5 g, 6.7 mol), water (115mL) and 65 g of palladium on activated carbon (10% on dry basis, ˜50%wet with water; Degussa type E101NE/W) under a stream of N₂ whilestirring via mechanical stirrer. After 24 and 48 hours, additionalportions of ammonium formate (132 g, 2.1 mol) were added each time. Oncereaction progression ceased, as monitored by anal. HPLC, Celite® (>500g) was added and the resulting thick suspension was filtered and thenthe collected solid was rinsed with methanol (˜500 mL). The filtrateswere combined and concentrated under reduced pressure until all methanolhad been removed. The resulting cloudy, biphasic solution was thendiluted with 1M phosphoric acid to a final volume of 1.5 to 2.0 L at pH2 and washed with dichloromethane (3×700 mL). The aqueous layer wasbasified to pH 12 using 40% aq. NaOH, and extracted with dichloromethane(3×700 mL). The combined organic layers were dried over MgSO₄, filtered,and concentrated by rotary evaporation, then high-vacuum leaving 52 g(70%) of the title intermediate, commonly N-Boc-endo-3-aminotropane, asa white to pale yellow solid. The isomer ratio of endo to. exo amine ofthe product was >99 based on ¹H-NMR analysis (>96% purity by analyticalHPLC). ¹H NMR (CDCl₃) δ (ppm) 4.2-4.0 (broad d, 2H, CHNBoc), 3.25 (t,1H, CHNH₂), 2.1-2.05 (m, 4H), 1.9 (m, 2H), 1.4 (s, 9H, (CH₃)₃OCON),1.2-1.1 (broad, 2H). (m/z): [M+H]⁺ calcd for C₁₂H₂₂N₂O₂) 227.18; found,227.2. Analytical HPLC (isocratic method; 2:98 (A:B) to 90:10 (A:B) over5 min): retention time=3.68 min.

Preparation 2 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid

First, acetone (228.2 mL, 3.11 mol) was added to a stirred suspension of2-aminophenylmethanol (255.2 g, 2.07 mol) and acetic acid (3.56 mL, 62mmol) in water (2 L) at room temperature. After 4 h, the suspension wascooled to 0° C. and stirred for an additional 2.5 h and then filtered.The solid was collected and washed with water and the wet solid cooledand dried by lyophilisation to yield2,2,-dimethyl-1,4-dihydro-2H-benzo[1,3]oxazine (332.2 g, 98%) as anoff-white solid. ¹H NMR (CDCl₃; 300 MHz): 1.48 (s, 6H, C(CH ₃)₂), 4.00(bs, 1H, NH), 4.86 (s, 2H, CH ₂), 6.66 (d, 1H, ArH), 6.81 (t, 1H, ArH),6.96 (d, 1H, ArH), 7.10 (t, 1H, ArH).

A solution of 2,2,-dimethyl-1,4-dihydro-2H-benzo[1,3]oxazine (125 g,0.77 mol) in THF (1 L) was filtered through a scintillation funnel andthen added dropwise via an addition funnel, over a period of 2.5 h, to astirred solution of 1.0 M LiAlH₄ in THF (800 mL) at 0° C. The reactionwas quenched by slow portionwise addition of Na₂SO₄.10H₂O (110 g), overa period of 1.5 h, at 0° C. The reaction mixture was stirred overnight,filtered and the solid salts were washed thoroughly with THF. Thefiltrate was concentrated under reduced pressure to yield2-isopropylaminophenylmethanol (120 g, 95%) as a yellow oil. ¹H NMR(CDCl₃; 300 MHz): 1.24 (d, 6H, CH(CH ₃)₂), 3.15 (bs, 1H, OH), 3.61(sept, 1H, CH(CH₃)₂), 4.57 (s, 2H, CH ₂), 6.59 (t, 1H, ArH), 6.65 (d,1H, ArH), 6.99 (d, 1H, ArH), 7.15 (t, 1H, ArH).

Manganese dioxide (85% 182.6 g, 1.79 mol) was added to a stirredsolution of 2-isopropylaminophenylmethanol (118 g, 0.71 mol) in toluene(800 mL) and the reaction mixture was heated to 117° C. for 4 h. Thereaction mixture was allowed to cool to room temperature overnight andthen filtered through a pad of Celite which was eluted with toluene. Thefiltrate was concentrated under reduced pressure to yield2-isopropylaminobenzaldehyde (105 g, 90%) as an orange oil. ¹H NMR(CDCl₃; 300 MHz): 1.28 (d, 6H, CH(CH ₃)₂), 3.76 (sept, 1H, CH(CH₃)₂),6.65 (t, 1H, ArH, 6.69 (d, 1H, ArH), 7.37 (d, 1H, ArH, 7.44 (t, 1H, ArH,9.79 (s, 1H, CHO).

2,2-Dimethyl-[1,3]dioxane-4,6-dione, commonly Meldrum's acid, (166.9 g,1.16 mol) was added to a stirred solution of2-isopropylaminobenzaldehyde (105 g, 0.64 mol), acetic acid (73.6 mL,1.29 mol) and ethylenediamine (43.0 mL, 0.64 mol) in methanol (1 L) at0° C. The reaction mixture was stirred for 1 h at 0° C. and then at roomtemperature overnight. The resulting suspension was filtered and thesolid washed with methanol and collected to yield the titleintermediate, 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid(146 g, 98%) as an off-white solid. ¹H NMR (CDCl₃; 300 MHz): 1.72 (d,6H, CH(CH ₃)₂), 5.50 (bs, 1H, CH(CH₃)₂), 7.44 (t, 1H, ArH), 7.75-7.77(m, 2H, ArH), 7.82 (d, 1H, ArH), 8.89 (s, 1H, CH).

Preparation 3 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-azabicyclo[3.2.1]oct-3-yl}amide

a. Preparation of(1S,3R,5R)-3-[1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-8-azabicyclo[3.2.1]octane-8-carboxylicacid tert-butyl ester

Thionyl chloride (36.6 mL, 0.52 mol) was added to a stirred suspensionof 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid (80 g, 0.35mol) in toluene (600 mL) at 85° C. and the reaction mixture then heatedto 95° C. for 2 h. The reaction mixture was cooled to room temperatureand then added over 25 min to a vigorously stirred biphasic solution of(1S,3R,5R)-3-amino-8-azabicyclo[3.2.1]octane-8-carboxylic acidtert-butyl ester (78.2 g, 0.35 mol) and sodium hydroxide (69.2 g, 1.73mol) in toluene/water (1:1) (1 L) at ° C. After 1 h, the layers wereallowed to separate and the organic phase concentrated under reducedpressure. The aqueous phase was washed with EtOAc (1 L) and then (500mL) and the combined organic extracts used to dissolve the concentratedorganic residue. This solution was washed with 1M H₃PO₄ (500 mL), sat.aq. NaHCO₃ (500 mL) and brine (500 mL), dried over MgSO₄, filtered andconcentrated under reduced pressure to yield the title intermediate(127.9 g, approx. 84%) as a yellow solid. ¹H NMR (CDCl₃): 1.47 (s, 9H),1.67 (d, 6H), 1.78-1.84 (m, 2H), 2.04-2.18 (m, 6H), 4.20-4.39 (m, 3H),5.65 (bs, 1H), 7.26 (dd. 1H), 7.63 (m, 2H), 7.75 (dd, 1H), 8.83 (s, 1H),10.63 (d, 1H).

b. Preparation of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylicacid {(1S,3R,5R)-8-azabicyclo[3.2.1]oct-3-yl}amide

TFA (300 mL) was added to a stirred solution of the product of theprevious step (127.9 g) in CH₂Cl₂ (600 mL) at 0° C. The reaction mixturewas warmed to room temperature and stirred for 1 h and then concentratedunder reduced pressure. The oily brown residue was then poured into avigorously stirred solution of ether (3 L) and a solid precipitateformed immediately. The suspension was stirred overnight and then thesolid collected by filtration and washed with ether to yield the titleintermediate as its trifluoroacetic acid salt (131.7 g, 86% over twosteps) as a light yellow solid. ¹H NMR (CDCl₃): 1.68 (d, 6H), 2.10 (d,2H), 2.33-2.39 (m, 4H), 2.44-2.61 (m, 2H), 4.08 (bs, 2H), 4.41 (m, 1H),5.57 (bs, 1H), 7.31 (m. 1H), 7.66 (m, 2H), 7.77 (d, 1H), 8.83 (s, 1H),9.38 (bd, 2H), 10.78 (d, 1H).

Preparation 43-hydroxy-3′-{[1-isopropyl-2-oxo-1,2-dihydroquinolin-3-yl)carbonyl]amino}spiro[azetidine-1,8′-(1S,3R,5R)-8-azabicyclo[3.2.1]octane

2-Bromomethyloxirane (10.72 mL, 129.5 mmol) was added to a stirredsolution of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-azabicyclo[3.2.1]oct-3-yl}amide trifluoroacetic acid salt(14.65 g, 43.2 mmol) in ethanol (150 mL) at room temperature. Thereaction mixture was stirred for 36 h, at which time a solid precipitateformed. The solid was collected by filtration and washed with ethanol(70 mL) to yield the title intermediate as the bromide salt (8.4 g).(m/z): [M]⁺ calcd for C₂₃H₃₀N₃O₃ 396.23; found, 396.5. Retention time(anal. HPLC: 2-50% MeCN/H₂O over 5 min)=4.13 min.

Preparation 53-methoxy-3′-{[1-isopropyl-2-oxo-1,2-dihydroquinolin-3-yl)carbonyl]amino}spiro[azetidine-1,8′-(1S,3R,5R)-8-azabicyclo[3.2.1]octane

Potassium-tert-butoxide (1.63 g, 14.5 mmol) was added to a stirredsuspension of3-hydroxy-3′-{[1-isopropyl-2-oxo-1,2-dihydroquinolin-3-yl)carbonyl]amino}spiro-[azetidine-1,8′-(1S,3R,5R)-8-azabicyclo[3.2.1]octanebromide (3.45 g, 7.25 mmol) in dichloromethane (100 mL) at roomtemperature. After 2 min, methyl iodide (0.477 mL, 7.61 mmol) was addedto the reaction mixture. After 30 min, water (2 mL) was added to quenchthe reaction and the reaction mixture concentrated under reducedpressure. The residue was dissolved in a minimal volume of aceticacid/water (1:1) and purified by preparative HPLC to yield the titleintermediate as a trifluoroacetic acid salt (2.1 g). (m/z): [M]⁺ calcdfor C₂₄H₃₂N₃O₃, 410.24; found 410.5. Retention time (anal. HPLC: 2-50%MeCN/H₂O over 5 min)=4.36 min.

Preparation 63-(methylaminocarbonyloxy)-3′-{[1-isopropyl-2-oxo-1,2-dihydroquinolin-3-yl-carbonyl]amino}spiro[azetidine-1,8′-(1S,3R,5R)-8-azabicyclo[3.2.1]octane]

Methyl isocyanate dissolved in 2.0 mL of DMF (125 mg/mL, 4.2 mmol) wasadded to a solution of3-hydroxy-3′-{[1-isopropyl-2-oxo-1,2-dihydroquinolin-3-yl-carbonyl]amino}spiro[azetidine-1,8′-(1S,3R,5R)-8-aza-bicyclo[3.2.1]octane](2.0 g, 4.2 mmol) and N,N-diisopropylethylamine (0.73 mL, 4.2 mmol) inDMF (40 mL). A catalytic amount of potassium tert-butoxide (1% byweight) was added and the mixture was stirred at room temperature for 5h. An additional 4.2 mmol of methyl isocyanate andN,N-diisopropylethylamine were added and the reaction went to completionafter stirring for an additional. 16 h. The reaction mixture wasconcentrated under vacuum and the resultant solid was used as a crudeproduct (2.1 g). (m/z): [M+H]⁺ calcd for C₂₅H₃₃N₄O₄, 453.25; found453.2. Retention time (anal. HPLC: 10-70% MeCN/H₂O over 6 min)=2.38 min.¹H NMR (CD₃OD): δ (ppm) 1.62 (d, 6H), 2.16 (m, 2H), 2.00 (br d, 3H),2.40-2.57 (m, 6H), 4.09 (br s, 1H), 4.17 (br s, 1H), 4.26 (q, 1H), 4.53(br s, 1H), 5.22 (br s, 1H), 7.00 (br m, 1H), 7.25 (t, 1H), 7.69 (m,1H), 7.80 (m, 2H), 8.69 (s, 1H), 10.90 (d, 1H).

Example 1 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

Piperazine N-methylsulfonamide/trifluoroacetic acid salt (1.23 g, 4.41mmol) was added to a stirred solution of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-azabicyclo[3.2.1]oct-3-yl}amide/trifluoroacetic acid salt(2.00 g, 4.41 mmol) and N,N-diisopropylethylamine (3.46 mL, 19.85 mmol)in methanol (50 mL). 1,3-Dibromopropanol (0.45 mL, 4.41 mmol) wassubsequently added and the reaction mixture was heated to 75° C. for 16h, at which point a further amount of piperazine sulfonamide/TFA (798mg, 2.87 mmol) and 1,3-dibromopropanol (0.29 mL, 2.87 mmol) were addedand the reaction mixture heated at 75° C. for a further 2 h. Thereaction mixture was concentrated in vacuo, diluted with 50% aqueousacetic acid (8 mL) and purified by preparative HPLC (5-32% gradient) toafford the title compound (770 mg) as a white solid. (m/z): [M+H]⁺ calcdfor C₂₈H₄₁N₅O₅S 560.29; found, 560.2. Retention time (anal. HPLC: 2-40%MeCN/H₂O over 6 min)=4.05 min. ¹H-NMR (CD₃OD): δ (ppm) 1.62 (d, 6H),2.16 (m, 2H), 2.40-2.57 (m, 6H), 2.89 (s, 3H), 3.16 (m, 4H), 3.38 (br s,4H), 3.51 (br s, 4H), 4.09 (br s, 1H), 4.17 (br s, 1H), 4.26 (q, 1H),4.53 (br s, 1H), 5.45 (br s, 1H), 7.31 (t, 1H), 7.69 (m, 1H), 7.80 (m,2H), 8.74 (s, 1H), 11.00 (d, 1H).

Example 2 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid((1S,3R,5R)-8-{2-hydroxy-3-[4-(propane-2-sulfonyl)piperazin-1-yl]-propyl}-8-azabicyclo[3.2.1]oct-3-yl)amide

Following the procedure of Example 1 with the substitution of piperazineN-isopropylsulfonamide for piperazine N-methylsulfonamide, the titlecompound was prepared. (m/z): [M+H]⁺ calcd for C₃₀H₄₅N₅O₅S, 588.32;found 588.4. Retention time (anal. HPLC: 5-75% MeCN/H₂O over 6 min)=2.16min. ¹H NMR (CD₃OD): δ (ppm) 1.22 (d, 6H), 1.62 (d, 6H), 2.16 (m, 2H),2.40-2.57 (m, 6H), 3.08 (m, 2H), 3.38 (br s, 4H), 3.51 (br s, 4H), 4.09(br s, 1H), 4.17 (br s, 1H), 4.26 (q, 1H), 4.41 (br s, 1H), 7.31 (t,1H), 7.69 (m, 1H), 7.80 (m, 2H).

Example 3 Alternative synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid((1S,3R,5R)-8-{2-hydroxy-3-[4-(propane-2-sulfonyl)-piperazin-1-yl]propyl}-8-azabicyclo[3.2.1]oct-3-yl)amide

Piperazine N-isopropylsulfonamide trifluoroacetic acid salt (128 mg,0.42 mmol) was dissolved in a solution of3-hydroxy-3′-{[1-isopropyl-2-oxo-1,2-dihydroquinolin-3-yl-carbonyl]amino}spiro[azetidine-1,8′-(1S,3R,5R)-8-aza-bicyclo[3.2.1]octane](100 mg, 0.21 mmol) and N,N-diisopropylethylamine (0.11 mL, 0.63 mmol)in ethanol (10 mL). The reaction mixture was shaken in a heating blockat 100° C. for 3 h. It was then concentrated under vacuum, diluted with50% aqueous acetic acid (7.5 mL) and purified by preparative HPLC (2-40%gradient) to afford the title compound (93 mg) as a white solid.

Example 4 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(1,1-dioxo-1λ⁶-thiomorpholin-4-yl)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

Following the procedure of Example 3 with the substitution ofthiomorpholine-1,1-dioxide for piperazine N-isopropylsulfonamide, thetitle compound was prepared. (m/z): [M+H]⁺ calcd for C₂₇H₃₈N₄O₅S,531.27; found 531.3. Retention time (anal. HPLC: 2-50% MeCN/H₂O over 6min)=3.60 min.

Example 5 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(4-acetylpiperazin-1-yl)-2-methoxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

N-Acetylpiperazine (0.16 mmol) was dissolved in a solution of3-methoxy-3′-{[1-isopropyl-2-oxo-1,2-dihydroquinolin-3-yl-carbonyl]amino}spiro[azetidine-1,8′-(1S,3R,5R)-8-azabicyclo[3.2.1]octane](42 mg, 0.08 mmol) and N,N-diisopropylethyl-amine (0.056 mL, 0.32 mmol)in ethanol (1 mL). The reaction mixture was shaken in a heating block at80° C. for 16 h. The reaction mixture was concentrated under vacuum,diluted with 50% aqueous acetic acid (1.5 mL) and purified bypreparative HPLC (5-32% gradient) to afford the title compound. (m/z):[M+H]⁺ calcd for C₃₀H₄₃N₅O₄, 538.34; found, 538.4. Retention time (anal.HPLC: 5-65% MeCN/H₂O over 4 min)=2.12 min.

Example 6 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(4-methanesulfonylmethanesulfonylpiperazin-1-yl)-2-methoxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

Following the procedure of Example 5 with the substitution of piperazineN-(1-methylsulfonyl)methanesulfonamide for N-acetylpiperazine, the titlecompound was prepared. (m/z): [M+H]⁺ calcd for C₃₀H₄₅N₅O₇S₂, 652.29;found, 652.2. Retention time (anal. HPLC: 5-65% MeCN/H₂O over 4min)=2.31 min.

Example 7 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(1,1-dioxo-1λ⁶-thiomorpholin-4-yl)-2-methoxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

Following the procedure of Example 5 with the substitution ofthiomorpholine-1,1-dioxide for N-acetylpiperazine, the title compoundwas prepared. (m/z): [M+H]⁺ calcd for C₂₈H₄₀N₄O₅S, 545.28; found 545.2.Retention time (anal. HPLC: 5-65% MeCN/H₂O over 4 min)=2.53 min.

Example 8 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(S)-2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)-propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

a. Preparation of(S)-1-chloro-3-(4-methylsulfonyl-1-piperazinyl)-2-propanol

(S)-Epichlorohydrin (48.0 mL, 0.612 mol) was added to a stirred solutionof piperazine N-methylsulfonamide (87.3 g, 0.532 mol) in ethanol (1.33L) at room temperature. The reaction mixture was stirred for 18 h andthe white solid precipitate which formed was collected by filtration andwashed with ethanol to afford(S)-1-chloro-3-(4-methylsulfonyl-1-piperazinyl)-2-propanol (107.76 g) asa white solid which was used without further purification. (m/z): [M+H]⁺calcd for C₈H₁₇ClN₂O₃S, 257.07; found, 257.2. ¹H-NMR (DMSO): δ (ppm)2.37 (dd, 1H), 2.45 (dd, 1H), 2.50-2.58 (m, 4H), 2.86 (s, 3H), 3.09 (m,4H), 3.55 (dd, 1H), 3.65 (dd, 1H), 3.84 (m, 1H), 5.09 (d, 1H).

b. Preparation of (S)-1-methylsulfonyl-4-(oxiranylmethyl)piperazine

Sodium hydroxide (22.15 g, 0.534 mol) was added to a vigorously stirredsolution of the product of the previous step (118.13 g, 0.461 mol) in80% THF in water (1500 mL) at 0° C. The reaction mixture was stirred for90 min and the layers were separated. The organic layer was concentratedunder vacuum and diluted with dichloromethane (1500 mL) and washed witha mixture of the previously separated aqueous layer and 1M NaOH (500mL). The organic layer was further washed with 1M NaOH (500 mL) andbrine (500 mL), dried (MgSO₄), filtered and concentrated under vacuum toyield the title intermediate (90.8 g) as a white crystalline solid. Theproduct was recrystallised from hot 1:1 mixture of EtOAc and hexane (800mL) to yield 43.33 g of pure epoxide. (m/z): [M+H]⁺ calcd forC₈H₁₆N₂O₃S, 221.10; found 221.3. ¹H-NMR (DMSO-d₆): δ (ppm) 2.22 (dd,1H), 2.45-2.60 (m, 5H), 2.69-2.75 (m, 2H), 2.87 (s, 3H), 3.02 (m, 1H),3.11 (m, 4H).

c. Synthesis of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(S)-2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

A suspension of(S)-1-methylsulfonyl-4-(oxiranylmethyl)piperazine (69.4g, 0.316 mol) in ethanol (980 mL) was added to1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-azabicyclo[3.2.1]oct-3-yl}amide (100 g, 0.295 mol) and thereaction mixture heated at 80° C. for 18.5 h. The reaction mixture wascooled to room temperature, and concentrated under vacuum. The foamysolid was suspended in a mixture of acetonitrile and water (860 mL/940mL), heated, and sonicated until it became homogenous. The solution wasfiltered while hot and the filtrate was allowed to cool to 5° C.Crystals were formed and collected by filtration to yield the titlecompound (122 g) as a white crystalline solid. (m/z): [M+H]⁺ calcd forC₂₈H₄₁N₅O₅S, 560.29; found 560.5. Retention time (anal. HPLC: 2-40%MeCN/H₂O over 6 min)=4.05 min. ¹H-NMR (CD₃OD, 400 MHz): δ (ppm) 1.68 (d,6H, CH(CH ₃)₂), 1.73-1.76 (br d, 2H), 2.10 (br s, 4H, 2×CHCH ₂), 2.28(m, 2H), 2.36 (dd, 1H), 2.42 (dd, 1H), 2.47-2.53 (m, 2H), 2.65 (m, 4H),2.85 (s, 3H, SO₂CH ₃), 3.24 (t, 4H, 2×CH ₂SO₂CH₃), 3.29 (br s, 1H, CHN),3.36 (br s, 1H, CHN), 3.86 (m, 1H, CHNH), 4.19 (t, 1H, CHOH), 5.50 (brs, 1H, CH(CH₃)₂), 7.34 (t, 1H, ArH), 7.72 (m, 1H, ArH), 7.83 (m, 2H,2×ArH), 8.76 (s, 1H, C═CH). ¹³C-NMR (CD₃OD, 100 MHz): δ (ppm) 19.8 (q,CH(CH₃)₂), 26.7, 26.8 (two t), 34.3 (q, SO₂ CH₃), 37.2 (t), 42.5 (d),46.9 (t), 54.3 (t), 58.3 (t), 60.2, 60.9 (two d), 63.6 (t), 68.5 (d),116.5 (d, CH(CH₃)₂), 121.7, 122.4 (two s), 124.1, 132.4, 133.9 (threed), 141.4 (s), 144.7 (d), 164.0, 164.4 (two s, 2×C═O).

Example 9 Synthesis of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)-propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

a. Preparation of(R)-1-chloro-3-(4-methylsulfonyl-1-piperazinyl)-2-propanol

(R)-Epichlorohydrin (3.10 mL, 39.5 mmol) was added to a stirred solutionof piperazine sulfonamide trifluoroacetic acid (10.0 g, 35.9 mmol) andN,N-diisopropyl-ethylamine (6.26 mL, 35.9 mmol) in ethanol (150 mL) atroom temperature. The reaction mixture was stirred for 18 h and afurther amount of (R)-epichlorohydrin was added (0.28 mL, 3.6 mmoL) andstirred for an additional 3 h. The reaction mixture was concentratedunder vacuum and the white solid was suspended in ethanol (150 mL) andstirred for 2 days. The solid was collected by filtration and washedwith cold ethanol to yield(R)-1-chloro-3-(4-methylsulfonyl-1-piperazinyl)-2-propanol (5.69 g) as awhite solid which was used without further purification. (m/z): [M+H]⁺calcd for C₈H₁₇ClN₂O₃S, 257.07; found 257.2. ¹H-NMR (DMSO-d₆): δ (ppm)2.37 (dd, 1H), 2.45 (dd, 1H), 2.50-2.58 (m, 4H), 2.86 (s, 3H), 3.09 (m,4H), 3.55 (dd, 1H), 3.65 (dd, 1H), 3.84 (m, 1H), 5.09 (d, 1H).

b. Preparation of (R)-1-methylsulfonyl-4-(oxiranylmethyl)piperazine

Sodium hydroxide (1.07 g, 26.7 mmol) was added to a vigorously stirredsolution of the product of the previous step (5.69 g, 22.2 mmol) in amixture of 80% THF in water (180 mL). The reaction mixture was stirredfor 35 min, concentrated under vacuum to approximately 50 mL by volumeand diluted with chloroform (200 mL) and washed with 1M NaOH (2×70 mL)and brine (70 mL). The organic layer was dried (MgSO₄), filtered andconcentrated under vacuum to yield the title intermediate (4.35 g) as awhite crystalline solid. The product was recrystallised from hotEtOAc/hexane (1:1; 800 mL) to yield pure epoxide (2.62 g). (m/z): [M+H]⁺calcd for C₈H₁₆N₂O₃S, 221.10; found, 221.3. ¹H-NMR (DMSO-d₆): δ (ppm)2.22 (dd, 1H), 2.45-2.60 (m, 5H), 2.69-2.75 (m, 2H), 2.87 (s, 3H), 3.02(m, 1H), 3.11 (m, 4H).

c. Synthesis of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide

1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-azabicyclo[3.2.1]oct-3-yl}amide (1.49 g, 4.38 mmol) wasadded to a stirred solution of(R)-1-methylsulfonyl-4-(oxiranylmethyl)piperazine (964 mg, 4.38 mmol) intoluene (20 mL) and the reaction mixture heated at 98° C. for 15 h. Thereaction mixture was cooled to room temperature, concentrated undervacuum, diluted with 50% aqueous acetic acid (12 mL) and purified bypreparative HPLC (5-30% gradient) to afford the title compound (492 mg)as a white solid. (m/z): [M+H]⁺ calcd for C₂₈H₄₁N₅O₅S, 560.29; found,560.2. Retention time (anal. HPLC: 2-40% MeCN/H₂O over 6 min)=4.05 min.¹H-NMR (CD₃OD): δ (ppm) 1.62 (d, 6H), 2.16 (m, 2H), 2.40-2.57 (m, 6H),2.89 (s, 3H), 3.16 (m, 4H), 3.38 (br s, 4H), 3.51 (br s, 4H), 4.09 (brs, 1H), 4.17 (br s, 1H), 4.26 (q, 1H), 4.53 (br s, 1H), 5.45 (br s, 1H),7.31 (t, 1H), 7.69 (m, 1H), 7.80 (m, 2H), 8.74 (s, 1H), 11.00 (d, 1H).

Example 10 Synthesis of methyl-carbamic acid2-{(1S,3R,5R)-3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-8-azabicyclo-[3.2.1]oct-8-yl}-1-(4-methanesulfonylpiperazin-1-ylmethyl)ethylester

Piperazine N-methylsulfonamide trifluoroacetic acid salt (0.20 mmol) wasadded to a solution of3-(methylaminocarbonyloxy)-3′-{[1-isopropyl-2-oxo-1,2-dihydroquinolin-3-yl-carbonyl]amino}spiro[azetidine-1,8′-(1S,3R,5R)-8-azabicyclo[3.2.1]octane](45.4 mg, 0.10 mmol) and N,N-diisopropylethylamine (0.087 mL, 0.50 mmol)in DMF (1 mL). The reaction mixture was shaken in a heating block at 85°C. for 16 h. The reaction mixture was concentrated under vacuum, dilutedwith 50% aqueous acetic acid (7.5 mL) and purified by preparative HPLC(2-50% gradient) to afford the title compound (24 mg) as a white solid.(m/z): [M+H]⁺ calcd for C₃₀H₄₄N₆O₆S, 617.31; found 617.2. Retention time(anal. HPLC: 2-50% MeCN/H₂O over 6 min)=3.82 min.

Example 11 Synthesis of methyl-carbamic acid1-(4-dimethylcarbamoyl-piperazin-1-ylmethyl)-2-{(1S,3R,5R)-3-[(1-isopropyl-2-oxo-1,2-dihydro-quinoline-3-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}ethylester

Following the procedure of Example 10 with the substitution of1-(dimethylcarbamoyl)piperazine for piperazine N-methylsulfonamide, thetitle compound was prepared. (m/z): [M+H]⁺ calcd for C₃₂H₄₇N₇O₅, 610.37;found 610.4. Retention time (anal. HPLC: 2-65% MeCN/H₂O over 4 min)=2.67min.

Example 12 Synthesis of methyl-carbamic acid1-[3-(acetylmethylamino)-pyrrolidin-1-ylmethyl]-2-{(1S,3R,5R)-3-[(1-isopropyl-2-oxo-1,2-dihydro-quinoline-3-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}ethylester

Following the procedure of Example 10 with the substitution of3-(N-acetyl-N-methylamino)pyrrolidine for piperazineN-methylsulfonamide, the title compound was prepared. (m/z): [M+H]⁺calcd for C₃₂H₄₆N₆O₅, 595.36; found 595.4. Retention time (anal. HPLC:5-65% MeCN/H₂O over 4 min)=2.63 min.

Example 13 Synthesis of methyl-carbamic acid1-(4-acetylpiperazin-1-ylmethyl)-2-{(1S,3R,5R))-3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}ethylester

Following the procedure of Example 10 with the substitution ofN-acetylpiperazine for piperazine N-methylsulfonamide, the titlecompound was prepared. (m/z): [M+H]⁺ calcd for C₃₁H₄₄N₆O₅, 581.35; found581.2. Retention time (anal. HPLC: 5-65% MeCN/H₂O over 4 min)=2.20 min.

Example 14 Synthesis of methyl-carbamic acid(R)-2-{(1S,3R,5R)₃-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-8-azabicyclo-[3.2.1]oct-8-yl}-1-(4-methanesulfonylpiperazin-1-ylmethyl)ethylester

Methyl isocyanate (41 mg, 7.1 mmol) was added to a solution of1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide(Example 9) (400 mg, 0.71 mmol) in toluene (10 mL). The reaction mixturewas capped and shaken in a heating block at 110° C. for 16 h. Thereaction mixture was concentrated under vacuum, diluted with 50% aqueousacetic acid (7.5 mL) and purified by preparative HPLC (5-10-40%gradient) to afford the title compound (110 mg) as a white solid. (m/z):[M+H]⁺ calcd for C₃₀H₄₄N₆O₆S, 617.31; found 617.4. Retention time (anal.HPLC: 10-70% MeCN/H₂O over 6 min)=2.40 min.

Example 15 Compounds of the Invention

Using the procedures of Examples 1-14 and variations thereof, thecompounds of Tables I to XXVIII were prepared and characterized by massspectrometry. In tables containing compounds prepared as purestereoisomers, the chirality at the carbon atom marked with an asteriskis indicated in the column headed by an asterisk. In the compounds ofTables I to XXVIII, the quinolinone-carboxamide group is in the endoconfiguration with respect to the azabicyclooctane group.

TABLE I

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R⁹ Formula calcd found 1 H OH H2-furanyl C₃₂H₄₅N₅O₅ 580.35 580.3 2 H OH H CH₃ C₂₉H₄₁N₅O₄ 524.33 524.4 3H OH CH₃ CH₃ C₃₀H₄₃N₅O₄ 538.34 538.4 4 H OCH₃ H CH₃ C₃₀H₄₃N₅O₄ 538.34538.4 5 H OC(O)N(CH₃)₂ H 2-furanyl C₃₅H₅₀N₆O₆ 651.39 651.4 6 HOC(O)N(CH₃)₂ H CH₃ C₃₂H₄₆N₆O₅ 595.36 595.4 7 H OC(O)N(CH₃)₂ H CH₃C₃₁H₄₄N₆O₅ 581.35 581.2 8 CH₃ OH H 2-furanyl C₃₃H₄₇N₅O₅ 594.37 594.4 9CH₃ OH H CH₃ C₃₀H₄₃N₅O₄ 538.34 538.4 10 F OH H CH₃ C₂₉H₄FN₅O₄ 542.32542.2 11 F OH H 2-furanyl C₃₂H₄₄FN₅O₅ 598.34 598.4

TABLE II

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R¹⁰ * Formula calcd found 1 HOH H CH₃ C₂₈H₄₁N₅O₅S 560.29 560.2 2 H OH H C₂H₅ C₂₉H₄₃N₅O₅S 574.31 574.23 H OH H CH₂cyclolohexyl C₃₄H₅₁N₅O₅S 642.37 642.4 4 H OH H CH(CH₃)₂C₃₀H₄₅N₅O₅S 588.32 588.4 5 H OCH₃ H CH₃ C₂₉H₄₃N₅O₅S 574.31 574.5 6 H OHH CH₂SO₂CH₃ C₂₉H₄₃N₅O₇S₂ 638.27 638.2 7 H OH H CH₃ R C₂₈H₄₁N₅O₅S 560.29560.5 8 H OH H CH₃ S C₂₈H₄₁N₅O₅S 560.29 560.5 9 Br OH H CH₃ SC₂₈H₄₀BrN₅O₅S 638.20 638.0 10 H OC(O)N(CH₃)₂ CH₃ CH₃ C₃₁H₄₆N₆O₆S 631.33631.2 11 H OCH₃ H CH₂SO₂CH₃ C₃₀H₄₅N₅O₇S₂ 652.29 652.2 12 H OH H CH₂CF₃C₂₉H₄₀F₃N₅O₅S 628.28 628.2 13 H OC(O)NHCH₃ H CH₃ C₃₀H₄₄N₆O₆S 617.31617.2 14 H OC(O)NHCH₃ H CH₃ S C₃₀H₄₄N₆O₆S 617.31 617.4 15 H OC(O)NHCH₃ HCH₃ R C₃₀H₄₄N₆O₆S 617.31 617.4 16 CH₃ OH H CH₃ C₂₉H₄₃N₅O₅S 574.31 574.417 F OH H CH₃ C₂₈H₄₀FN₅O₅S 578.28 578.2 18 H CH₂OH H CH₃ C₂₉H₄₃N₅O₅S574.31 574.5

TABLE III

[M + Molecular [M + H]⁺ H]⁺ No. R¹ R³ R⁴ Formula calcd found 1 H OH HC₂₇H₃₈N₄O₅S 531.27 531.3 2 H OCH₃ H C₂₈H₄₀N₄O₅S 545.28 545.2 3 HOC(O)N(CH₃)₂ H C₃₀H₄₃N₅O₆S 602.30 602.2 4 H OC(O)NHCH₃ H C₃₂H₄₆N₆O₅595.36 595.4 5 CH₃ OH H C₂₈H₄₀N₄O₅S 545.28 545.2 6 F OCH₃ H C₂₇H₃₇FN₄O₅S549.26 549.2 7 H CH₂OH H C₂₈H₄₀N₄O₅S 545.28 545.3

TABLE IV

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R¹² Formula calcd found 1 H OHH C₂H₅ C₃₀H₄₃N₅O₅ 554.34 554.3 2 H OH H CH₃ C₂₉H₄₁N₅O₅ 540.32 540.4 3 HOH CH₃ CH₃ C₃₀H₄₃N₅O₅ 554.34 554.2 4 H OCH₃ H CH₃ C₃₀H₄₃N₅O₅ 554.34554.2 5 H OC(O)N(CH₃)₂ H CH₃ C₃₂H₄₆N₆O₆ 611.36 611.4 6 H OC(O)NHCH₃ HCH₃ C₃₁H₄₄N₆O₆ 597.34 597.2

TABLE V

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R¹³ R¹⁴ Formula calcd found 1 HOH H CH₃ CH₃ C₃₀H₄₄N₆O₄ 553.35 553.2 2 H OCH₃ H CH₃ CH₃ C₃₁H₄₆N₆O₄567.37 567.4 3 H OC(O)N(CH₃)₂ H CH₃ CH₃ C₃₃H₄₉N₇O₅ 624.39 624.4 4 HOC(O)NHCH₃ H CH₃ CH₃ C₃₂H₄₇N₇O₅ 610.37 610.4 5 H OC(O)NHCH₃ H CH₃ HC₃₁H₄₅N₇O₅ 596.36 596.2 6 H OH H H H C₂₈H₄₀N₆O₄ 525.32 525.2 7 H OH HCH₃ H C₂₉H₄₂N₆O₄ 539.34 539.2 8 CH₃ OH H CH₃ CH₃ C₃₁H₄₆N₆O₄ 567.37 567.49 F OH H CH₃ CH₃ C₃₀H₄₃FN₆O₄ 571.34 571.4 10 F OH H CH₃ H C₂₉H₄₁FN₆O₄557.31 557.2

TABLE VI

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R¹³ R¹⁴ Formula calcd found 1 HOH H CH₃ CH₃ C₂₉H₄₄N₆O₅S 589.32 589.4 2 H OCH₃ H CH₃ CH₃ C₃₀H₄₆N₆O₅S603.34 603.4 3 H OC(O)N(CH₃)₂ H CH₃ CH₃ C₃₂H₄₉N₇O₆S 660.36 660.2 4 HOC(O)NHCH₃ H CH₃ CH₃ C₃₁H₄₇N₇O₆S 646.34 646.4 5 CH₃ OH H CH₃ CH₃C₃₀H₄₆N₆O₅S 603.34 603.4 6 F OCH₃ H CH₃ CH₃ C₂₉H₄₃FN₆O₅S 607.31 607.2

TABLE VII

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R⁸ R⁹ * Formula calcd found 1 HOH H CH₃ CH₃ C₃₀H₄₃N₅O₄ 538.34 538.3 2 H OH H H CH₃ R C₂₉H₄₀N₅O₄ 524.33524.4 3 H OH H C₂H₅ CH₃ C₃₁H₄₅N₅O₄ 552.36 552.4 4 H OCH₃ H CH₃ CH₃C₃₁H₄₅N₅O₄ 552.36 552.3 5 H OH H H CF₃ R C₂₉H₃₈F₃N₅O₄ 578.30 578.2 6 HOH H H CF₃ C₂₉H₃₈F₃N₅O₄ 578.30 578.2 7 H OH H H CH₃ C₂₉H₄₁N₅O₄ 524.33524.4 8 H OH CH₃ C₂H₅ CH₃ C₃₂H₄₇N₅O₄ 566.37 566.4 9 H OH H H CH(OH)CH₃C₃₀H₄₃N₅O₅ 554.34 554.4 10 H OCH₃ H H CF₃ S C₃₀H₄₀F₃N₅O₄ 592.31 592.2 11H OC(O)N(CH₃)₂ H C₂H₅ CH₃ C₃₄H₅₀N₆O₅ 623.39 623.4 12 H OC(O)NHCH₃ H CH₃CH₃ C₃₂H₄₆N₆O₅ 595.36 595.4 13 CH₃ OH H CH₃ CH₃ C₃₁H₄₅N₅O₄ 552.36 552.414 CH₃ OH H H CH₃ C₃₀H₄₃N₅O₄ 538.34 538.4 15 F OH H CH₃ CH₃ C₃₀H₄₂FN₅O₄556.33 556.2

TABLE VIII

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R^(6a) R⁸ R¹⁰ * Formula calcdfound 1 H OH H H H CH₃ R C₂₈H₄₁N₅O₅S 560.29 560.2 2 H OH H H H C₂H₅ RC₂₉H₄₃N₅O₅S 574.31 574.3 3 H OH H H H CH(CH₃)₂ R C₃₀H₄₅N₅O₅S 588.32588.2 4 H OH H H H CH₂SO₂CH₃ R C₂₉H₄₃N₅O₇S₂ 638.27 638.2 5 H OH H H HCH₂cyclohexyl R C₃₄H₅₁N₅O₅S 642.37 642.3 6 H OH H H CH₃ CH₃ C₂₉H₄₃N₅O₅S574.31 574.2 7 H OH H H H CH₃ C₂₈H₄₁N₅O₅S 560.29 560.2 8 H OCH₃ H H CH₃CH₃ C₃₀H₄₅N₅O₅S 588.32 588.4 9 H OC(O)N(CH₃)₂ H H CH₃ CH₃ C₃₂H₄₈N₆O₆S645.35 645.4 10 H OH H (S)- CH₃ CH₃ S C₂₉H₄₃N₅O₆S 590.30 590.2 OH

TABLE IX

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R^(6a), R^(7a) R¹³ R¹⁴ *Formula calcd found 1 H OH H H, H H H R C₂₈H₃₉N₅O₄ 510.31 510.3 2 H OH HH, H CH₃ CH₃ S C₃₀H₄₃N₅O₄ 538.34 538.3 3 H OH H F, F C₂H₅ C₂H₅ SC₃₂H₄₅F₂N₅O₄ 602.35 602.4 4 H OH H H, H H H S C₂₈H₃₉N₅O₄ 510.31 510.4 5H OCH₃ H H, H H H R C₂₉H₄₁N₅O₄ 524.33 524.5 6 H OH CH₃ H, H H H RC₂₉H₄₁N₅O₄ 524.33 524.4 7 H OH CH₃ H, H CH₃ CH₃ R C₃₁H₄₅N₅O₄ 552.36552.4 8 H OC(O)N(CH₃)₂ H H, H H H R C₃₁H₄₄N₆O₅ 581.35 581.4 9 HOC(O)N(CH₃)₂ H H, H CH₃ CH₃ S C₃₃H₄₈N₆O₅ 609.38 609.4 10 H OC(O)NHCH₃ HH, H CH₃ CH₃ R C₃₂H₄₆N₆O₅ 595.36 595.4 11 CH₃ OH H H, H H H S C₂₉H₄₁N₅O₄524.33 524.4 12 CH₃ OH H H, H H H R C₂₉H₄₁N₅O₄ 524.33 524.4 13 F OH H H,H H H R C₂₈H₃₈FN₅O₄ 528.30 528.2 14 F OH H H, H H H S C₂₈H₃₈FN₅O₄ 528.30528.2

TABLE X

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R^(6a) X * Formula calcd found 1 HOH H NHC(O)OCH₃ C₂₉H₄₁N₅O₅ 540.32 540.4 2 H OH H N(CH₃)C(O)OCH₃C₃₀H₄₃N₅O₅ 554.34 554.2 3 H OH (S) OH N(CH₃)C(O)OCH₃ S C₃₀H₄₃N₅O₆ 570.33570.4 4 H OH H OC(O)N(CH₃)₂ C₃₀H₄₃N₅O₅ 554.34 554.2 5 F OH HOC(O)N(CH₃)₂ C₃₀H₄₂FN₅O₅ 572.33 572.2 6 H OH H NHSO₂N(CH₃)₂ RC₂₉H₄₄N₆O₅S 589.32 589.3 7 H OH H N(CH₃)SO₂N(CH₃)₂ C₃₀H₄₆N₆O₅S 603.34603.2 8 H OH H NHSO₂N(CH₃)₂ C₂₉H₄₄N₆O₅S 589.32 589.2 9 H OH HNHC(O)N(CH₃)₂ C₃₀H₄₄N₆O₄ 553.35 553.4 10 H OH H N(CH₃)C(O)N(CH₃)₂C₃₀H₄₄N₆O₄ 553.35 553.4 11 H OCH₃ H N(CH₃)C(O)N(CH₃)₂ C₃₂H₄₈N₆O₄ 581.38581.4 12 H OH (S) OH N(CH₃)C(O)N(CH₃)₂ S C₃₁H₄₆N₆O₅ 583.36 583.4 13 CH₃OH H N(CH₃)C(O)N(CH₃)₂ C₃₂H₄₈N₆O₄ 581.38 581.4 14 F OH HN(CH₃)C(O)N(CH₃)₂ C₃₁H₄₅FN₆O₄ 585.36 585.4 15 H OH H NH₂ S C₂₉H₄₃N₅O₃510.35 510.3 16 H OH H NH₂ R C₂₉H₄₃N₅O₃ 510.35 510.3 17 H OH H2-pyridinyl C₃₂H₄₁N₅O₃ 544.33 544.4 18 H OH H 1,1-dioxo- C₃₀H₄₃N₅O₅S586.31 586.3 isothiazolidin-2-yl

TABLE XI

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R¹⁵ * Formula calcd found 1 HOH H H S C₂₈H₄₀N₄O₄ 497.31 497.3 2 H OH H CH₃ S C₂₉H₄₂N₄O₄ 511.33 511.33 H OC(O)N(CH₃)₂ H H S C₃₁H₄₅N₅O₅ 568.35 568.4 4 H OC(O)N(CH₃)₂ H CH₃ SC₃₂H₄₇N₅O₅ 582.37 582.4

TABLE XII

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ X Formula calcd found 1 H OH HNHSO₂CH₃ C₂₉H₄₃N₅O₅S 574.31 574.3 2 H OH H NHSO₂C₂H₅ C₃₀H₄₅N₅O₅S 588.32588.3 3 H OH H NHSO₂CH₂SO₂CH₃ C₃₀H₄₅N₅O₇S₂ 652.29 652.2 4 H OH HNHC(O)OCH₃ C₃₀H₄₃N₅O₅ 554.34 554.4 5 F OH H NHSO₂N(CH₃)₂ C₃₀H₄₆N₆O₅S603.34 603.3 6 H OH H NHC(O)N(CH₃)₂ C₃₁H₄₆N₆O₄ 567.37 567.4 7 H OH HNHSO₂N(CH₃)₂ C₃₁H₄₈N₆O₅S 617.35 617.4

TABLE XIII

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R^(6a), R^(7a) R¹³ R¹⁴ Formulacalcd found 1 H OH H F, F C₂H₅ C₂H₅ C₃₃H₄₇F₂N₅O₄ 616.37 616.4 2 H OH HH, H H H C₂₉H₄₁N₅O₄ 524.33 524.2 3 H OCH₃ H H, H H H C₃₀H₄₃N₅O₄ 538.34538.4 4 H OC(O)N(CH₃)₂ H H, H C₂H₅ C₂H₅ C₃₆H₅₄N₆O₅ 651.43 651.4 5 HOC(O)N(CH₃)₂ H H, H H H C₃₂H₄₆N₆O₅ 595.36 595.4 6 CH₃ OH H H, H H HC₃₀H₄₃N₅O₄ 538.34 538.4 7 F OH H H, H H H C₂₉H₄₀FN₅O₄ 542.32 542.2 8 HCH₂OH H H, H H H C₃₀H₄₃N₅O₄

TABLE XIV

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R⁸ R⁹ Formula calcd found 1 HOH H H CH₃ C₃₀H₄₃N₅O₄ 538.34 538.4 2 H OH H CH₃ CH₃ C₃₁H₄₅N₅O₄ 552.36552.4 3 H OCH₃ H CH₃ CH₃ C₃₂H₄₇N₅O₄ 566.37 566.4 4 H OC(O)N(CH₃)₂ H CH₃CH₃ C₃₄H₅₀N₆O₅ 623.39 623.4 5 CH₃ OH H CH₃ CH₃ C₃₂H₄₇N₅O₄ 566.37 566.4 6F OH H CH₃ CH₃ C₃₁H₄₄FN₅O₄ 570.35 570.4

TABLE XV

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R⁸ R¹⁰ * Formula calcd found 1H OH H H CH₃ S C₂₉H₄₃N₅O₅S 574.31 574.2 2 H OH H H C₂H₅ S C₃₀H₄₅N₅O₅S588.32 588.3 3 H OH H H CH₂SO₂CH₃ S C₃₀H₄₅N₅O₇S₂ 652.29 652.2 4 H OH HCH₃ CH₃ C₃₀H₄₅N₅O₅S 588.32 588.4 5 H OH H H CH(CH₃)₂ S C₃₁H₄₇N₅O₅S602.34 602.2 6 CH₃ OH H CH₃ CH₃ C₃₁H₄₇N₅O₅S 602.34 602.4 7 F OH H CH₃CH₃ C₃₀H₄₄FN₅O₅S 606.31 606.2

TABLE XVI

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ X * Formula calcd found 1 H OHH N(CH₃)SO₂N(CH₃)₂ S C₃₀H₄₆N₆O₅S 603.34 603.3 2 H OH H NHSO₂N(CH₃)₂C₃₁H₄₈N₆O₅S 617.35 617.4 3 H OH H NHC(O)N(CH₃)₂ C₃₁H₄₆N₆O₄ 567.37 567.44 H OH H N(CH₃)C(O)N(CH₃)₂ C₃₂H₄₈N₆O₄ 581.38 581.4 5 H OH H NHC(O)OCH₃C₃₀H₄₃N₅O₅ 554.34 554.4 6 H OH H N(CH₃)C(O)OCH₃ C₃₁H₄₅N₅O₅ 568.35 568.47 F OH H N(CH₃)C(O)OCH₃ C₃₁H₄₄FN₅O₅ 586.34 586.4 8 H OH H OC(O)N(CH₃)₂C₃₁H₄₅N₅O₅ 568.35 568.4 9 H OCH₃ H OC(O)N(CH₃)₂ C₃₂H₄₇N₅O₄ 566.37 566.410 CH₃ OH H OC(O)N(CH₃)₂ C₃₂H₄₇N₅O₅ 582.37 582.4

TABLE XVII

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R^(6a) R⁸ R¹⁰ Formula calcdfound 1 H OH H H H CH₃ C₃₀H₄₅N₅O₅S 588.78 588.3 2 H OH H OH H CH₃C₃₀H₄₅N₅O₆S 604.32 604.2 3 H OH H H CH₃ CH₃ C₃₁H₄₇N₅O₅S 602.34 602.3 4 HOC(O)N(CH₃)₂ H H H CH₃ C₃₃H₅₀N₆O₆S 659.36 659.4 5 H OC(O)NHCH₃ H H H CH₃C₃₂H₄₈N₆O₆S 645.35 645.4 6 CH₃ OH H H H CH₃ C₃₁H₄₇N₅O₅S 602.34 602.4 7 FOH H H H CH₃ C₃₀H₄₄FN₅O₅S 606.31 606.2

TABLE XVIII

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ X Formula calcd found 1 H OH HOH C₂₈H₄₀N₄O₄ 497.31 497.3 2 H OH H pyrrolidin-1-yl- C₃₂H₄₅N₅O₄ 564.36564.3 2-one 3 H OH H C(O)NH₂ C₂₉H₄₁N₅O₄ 524.33 524.3 4 H OH Hpyrrolidin-1-yl C₃₂H₄₇N₅O₃ 550.38 550.4 5 H OH H N(CH₃)SO₂CH₃C₃₀H₄₅N₅O₅S 588.32 588.3 6 H OH H NHSO₂CH₃ C₂₈H₄₁N₅O₅S 560.29 560.5 7 HOH H (CH₂)₂OH C₃₀H₄₄N₄O₄ 525.35 525.4 8 H OC(O)N(CH₃)₂ H OH C₃₁H₄₅N₅O₅568.35 568.4 9 H OC(O)N(CH₃)₂ H pyrrolidin-1-yl- C₃₅H₅₀N₆O₅ 635.39 635.42-one 10 H CH₂OH H OH C₂₉H₄₂N₄O₄ 511.33 511.5 11 H OH₂OH H N(CH₃)SO₂CH₃C₃₁H₄₇N₅O₅S 602.34 602.8

TABLE XIX

[M + [M + Molecular H]⁺ H]⁺ No. R¹ R³ R⁴ X′ Formula calcd found 1 H OH H(CH₂)₂OH C₃₀H₄₄N₄O₄ 525.35 525.4 2 H OH H CH₂OH C₂₉H₄₂N₄O₄ 511.33 511.43 H OH H C(O)NH₂ C₂₉H₄₁N₅O₄ 524.33 525.2 4 H OC(O)NHCH₃ H C(O)NH₂C₃₁H₄₄N₆O₅ 581.35 581.4 5 H OC(O)N(CH₃)₂ H CH₂OH C₃₂H₄₇N₅O₅ 582.37 582.4

TABLE XX

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R¹⁶ Formula calcd found 1 H OHH (CH₂)₂OH C₂₉H₄₃N₅O₄ 526.34 526.3 2 H OH H (CH₂)₂O(CH₂)₂OH C₃₁H₄₇N₅O₅570.37 570.3 3 H OH H CH₂C(O)-4-morpholinyl C₃₃H₄₈N₆O 609.38 609.4 4 HOH H 4-pyridinyl C₃₂H₄₂N₆O₃ 559.34 559.3 5 H OH H CH₂-4-pyridinylC₃₃H₄₄H₆O₃ 573.36 573.3 6 H OH H CH₂-2-tetrahydrofuranyl C₃₂H₄₇N₅O₄566.37 566.3 7 H OH H (CH₂)₂-4-morpholinyl C₃₃H₅₀N₆O₄ 595.40 595.4 8 HOH H CH₂C(O)N(CH₃)₂ C₃₁H₄₆N₆O₄ 567.37 567.3 9 H OH H 2-pyridinylC₃₂H₄₂N₆O₃ 559.34 559.4

TABLE XXI

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R⁵ X′ * Formula calcd found 1 HOH H CH₃ NSO₂C₂H₅ C₃₀H₄₅N₅O₅S 588.32 588.2 2 H OH H CH₃ NSO₂CH₃C₂₉H₄₃N₅O₅S 574.31 574.2 3 H OH H CH₃ NC(O)OCH₃ C₃₀H₄₃N₅O₅ 554.34 554.44 H OH H CH₃ NC(O)N(CH₃)₂ C₃₁H₄₆N₆O₄ 567.37 567.6 5 H OCH₃ H CH₃NC(O)N(CH₃)₂ C₃₂H₄₈N₆O₄ 581.38 581.4 6 H OH H CH₃ NC(O)CH₃ C₃₀H₄₃N₅O₄538.34 538.2 7 H OH H CH₃ NSO₂N(CH₃)₂ C₃₀H₄₆N₆O₅S 603.34 603.2 8 HOC(O)NHCH₃ H CH₃ NC(O)CH₃ C₃₂H₄₆N₆O₆ 611.36 611.4 9 CH₃ OH H CH₃NSO₂N(CH₃)₂ C₃₁H₄₅N₅O₄ 552.36 552.4 10 CH₃ OH H CH₃ NSO₂CH₃ RC₃₀H₄₅N₅O₅S 588.32 588.4 11 CH₃ OH H CH₃ NSO₂CH₃ C₃₀H₄₅N₅O₅S 588.32588.4

TABLE XXII

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R⁵ * Formula calcd found 1 H OHH CH₃ C₂₈H₄₀N₄O₅S 545.28 545.2 2 H OCH₃ H CH₃ C₂₉H₄₂N₄O₅S 559.30 559.2 3H OC(O)N(CH₃)₂ H CH₃ C₃₁H₄₅N₅O₆S 616.32 616.2 4 H OH H CH₂CH₂OHC₂₉H₄₂N₄O₆S 575.29 575.2 5 H OH H CH₃ R C₂₉H₄₃N₅O₅S 574.31 574.2 6 CH₃OH H CH₃ C₂₉H₄₂N₄O₅S 559.30 559.2 7 F OH H CH₃ C₂₈H₃₉FN₄O₅S 563.27 563.2

TABLE XXIII

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R⁵ V Formula calcd found 1 H OHH CH₃ C(O)CH₃ C₃₁H₄₅N₅O₄ 552.36 552.4 2 H OH H CH₃ C(O)OCH3 C₃₁H₄₅N₅O₅568.35 568.4 3 H OH H CH₃ SO₂CH₃ C₃₀H₄₅N₅O₅S 588.32 588.4 4 H OH H CH₃C(O)N(CH₃)₂ C₃₂H₄₈N₆O₄ 581.38 581.4 5 H OH H CH₃ SO₂N(CH₃)₂ C₃₁H₄₈N₆O₅S617.35 617.4

TABLE XXIV

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R⁵ Formula calcd found 1 H OH HCH₃ C₂₉H₄₂N₄O₅S 559.30 559.2 2 CH₃ OH H CH₃ C₃₀H₄₄N₄O₅S 573.31 573.4 3 FOH H CH₃ C₂₉H₄₁FN₄O₅S 577.29 577.2

TABLE XXV

[M + Molecular [M + H]⁺ H]⁺ No. R¹ R³ X′ Formula calcd found 1 H OHNSO₂CH₃ C₂₉H₄₃N₅O₅S 574.31 574.7 2 H OH NC(O)CH₃ C₃₀H₄₃N₅O₄ 538.34 538.23 CH₃ OH NC(O)CH₃ C₃₁H₄₅N₅O₄ 552.36 552.4 4 CH₃ OH NSO₂CH₃ C₃₀H₄₅N₅O₅S588.32 588.4 5 F OH NC(O)CH₃ C₃₀H₄₂FN₅O₄ 556.33 556.4 6 F OH NSO₂CH₃C₂₉H₄₂FN₅O₅S 592.30 592.2

TABLE XXVI

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ R⁵ Z Formula calcd found 1 H OHH CH₃ (CH₂)₃NHSO₂—CH₃ C₂₈H₄₃N₅O₅S 562.31 562.3 2 H OH H CH₃ CH₂CH₂OHC₂₆H₃₈N₄O₄ 471.30 471.3 3 H OH H CH₃ (CH₂)₂N(CH₃)₂ C₂₈H₄₃N₅O₃ 498.35498.3 4 H OH H CH₃ CH₂C(O)N—(CH₃)₂ C₂₈H₄₁N₅O₄ 512.33 512.3 5 H OH H CH₃(CH₂)₂-2- C₃₁H₄₁N₅O₃ 532.33 532.3 pyridinyl 6 H OH H CH₃ (CH₂)₂CNC₂₇H₃₇N₅O₃ 480.30 480.2 7 H OH H CH₃ CH₂C(O)NH₂ C₂₆H₃₇N₅O₄ 484.29 484.28 H OH H CH₂CH₂OH CH₂CH₂OH C₂₇H₄₀N₄O₅ 501.31 501.4 9 H OH H CH₂CH₂OCH₃CH₂CH₂OCH₃ C₂₉H₄₄N₄O₅ 529.34 529.4 10 H OH H CH₂CH₃ CH₂-4-pyridinylC₃₁H₄₁N₅O₃ 532.33 532.4 11 H OH H CH₃ (CH₂)₂-3-indolyl C₃₄H₄₃N₅O₃ 570.35570.4 12 H OH H CH₃ (CH₂)₂SO₂CH₃ C₂₇H₄₀N₄O₅S 533.28 533.2 13 H OH H CH₃CH₂CN C₂₇H₃₆N₄O₃ 465.29 465.2 14 H OH H CH₃ (CH₂)₂OCH₃ C₂₇H₄₀N₄O₄ 485.31485.2 15 H OH H CH₃ CH₂CN C₂₅H₃₃N₅O₃ 452.27 452.0 16 H OH H CH₂CN CH₂CNC₂₇H₃₄N₆O₃ 491.28 491.4 17 H OH H CH₃ (CH₂)₂CN C₂₈H₃₉N₅O₃ 494.32 494.318 H OH H CH₃ (CH₂)₂N(CH₃)—C(O)N(CH₃)₂ C₃₀H₄₆N₆O₄ 555.37 555.4 19 H OH HCH₃ (CH₂)₂N(CH₃)—C(O)OCH₃ C₂₉H₄₁N₅O₅ 542.34 542.4 20 H OH H CH₂CH₂OH(CH₂)₂N(CH₃)—SO₂CH₃ C₂₉H₄₄N₅O₆S 592.32 592.4 21 H OH H CH₂CH₂OCH₃(CH₂)₂N(CH₃)—SO₂CH₃ C₃₀H₄₇N₅O₆S 606.33 606.4 22 H OH H CH₃(CH₂)₂NHSO₂—CH₃ C₂₇H₄₁N₅O₅S 548.29 548.4 23 H OH H H (CH₂)₂NHSO₂—CH₃C₂₆H₃₉N₅O₅S 534.28 534.2 24 H OH H CH₃ (CH₂)₂N(CH₃)—SO₂CH₃ C₂₈H₄₃N₅O₅S562.31 562.2 25 H OH H H (S)-CH₂-2- C₂₈H₄₀N₄O₄ 497.31 497.2tetrahydrofuranyl 26 H OH H H (CH₂)₂OCH₃ C₂₆H₃₈N₄O₄ 471.30 471.2 27 H OHH H CH₂CF₃ C₂₅H₃₃F₃N₄O₃ 495.26 495.2 28 H OH H H (CH₂)₃-1- C₂₉H₄₀N₆O₃521.33 521.2 imidazolyl 29 H OH H H (CH₂)₂SCH₂CH₃ C₂₇H₄₀N₄O₃S 501.29501.2 30 H OH H H CH₂CN C₂₆H₃₄N₄O₃ 451.27 451.2 31 H OH H H(CH₂)₂-3-indolyl C₃₃H₄₁N₅O₃ 556.33 556.2 32 H OH H H (CH₂)₂OCH₂CH₂OHC₂₇H₄₀N₄O₅ 501.31 501.2 33 H OH H H CH₂CH(OH)CH₂OH C₂₆H₃₈N₄O₅ 487.29487.2 34 H OH H H (S)- C₂₇H₃₈N₄O₆ 515.29 515.2 CH[(CH₂)₂OH]—C(O)OH 35 HOH H H (S)- C₂₆H₃₆N₄O₆ 501.27 501.2 CH[CH₂OH]C(O)OH 36 H OH H H (CH₂)₃-C₃₀H₄₃N₅O₄ 538.34 538.2 pyrrolidin-1-yl- 2-one 37 H OH H H C(CH₃)(CH₂OH)C₂₇H₄₀N₄O₅ 501.31 501.2 (CH₂OH) 38 H OH H H (R)-CH₂-2- C₂₈H₄₀N₄O₄ 497.31497.2 tetrahydrofuranyl 39 H OH H H (R)- C₂₇H₃₈N₄O₆ 515.29 515.2CH[(CH₂)₂OH]—C(O)OH 40 H OH H CH₃ (CH₂)₂N(CH₃)—SO₂N(CH₃)₂ C₂₉H₄₆N₆O₅S591.34 591.2 41 H OH CH₃ CH₃ CH₂CH₂OCH₃ C₂₈H₄₂N₄O₄ 499.33 499.4 42 H OHCH₃ CH₃ (CH₂)₂NHSO₂—CH₃ C₂₈H₄₃N₅O₅S 562.31 562.2 43 H OCH₃ H CH₂CH₂OH(CH₂)₂N(CH₃)—SO₂CH₃ C₃₀H₄₇N₅O₆S 606.33 606.2 44 H OCH₃ H CH₃CH₂C(O)N—(CH₃)₂ C₂₉H₄₃N₅O₄ 526.34 526.4 45 H OCH₃ H CH₂CH₂OCH₃CH₂CH₂OCH₃ C₃₀H₄₆N₄O₅ 543.36 543.4 46 H (a) H CH₃ CH₂C(O)N—(CH₃)₂C₃₁H₄₆N₆O₅ 583.36 583.4 47 H (a) H CH₂CH₂OCH₃ CH₂CH₂OCH₃ C₃₂H₄₉N₅O₆600.38 600.4 48 H (a) H CH₃ CH₂CH₂OH C₂₉H₄₃N₅O₅ 542.34 542.2 49 H (a) HCH₃ CH₂CH₂OCH₃ C₃₀H₄₅N₅O₅ 556.35 556.4 50 H OH H CH₂CH₂OH (CH₂)₂SO₂CH₃C₂₈H₄₂N₄O₆S 563.29 563.2 51 H OH H CH₂CH₂OCH₃ (CH₂)₂SO₂CH₃ C₂₉H₄₄N₄O₆S577.31 577.2 52 H (b) H CH₂CH₂OCH₃ (CH₂)₂SO₂CH₃ C₃₁H₄₇N₅O₇S 634.33 634.453 H (b) H CH₃ (CH₂)₂SO₂CH₃ C₂₉H₄₃N₅O₆S 590.30 590.2 54 H (S)-OH H CH₂CNCH₂CN C₃₁H₄₄N₆O₅ 581.35 581.2 55 H OH H CH₃ (CH₂)₂-1,1-dioxo-C₂₉H₄₃N₅O₅S 574.31 574.3 isothiazolidin-2-yl 56 CH₃ OH H CH₃(CH₂)₂N(CH₃)C(O)OCH₃ C₃₀H₄₅N₅O₅ 556.35 556.4 57 CH₃ OH H CH₃(CH₂)₂NHSO₂CH₃ C₂₈H₄₃N₅O₅S 562.31 562.2 58 CH₃ OH H CH₃(CH₂)₂N(CH₃)—SO₂CH₃ C₂₉H₄₅N₅O₅S 576.32 576.4 59 CH₃ OH H CH₂CH₂OCH₃(CH₂)₂N(CH₃)—SO₂CH₃ C₃₁H₄₉N₅O₆S 620.35 620.4 60 CH₃ OH H CH₂CH₂OH(CH₂)₂SO₂CH₃ C₂₉H₄₄N₄O₆S 577.31 577.2 61 CH₃ OH H CH₃ (CH₂)₂-1,1-dioxo-C₃₀H₄₅N₅O₅S 588.32 588.4 isothiazolidin-2-yl 62 F OH H CH₃CH₂C(O)N—(CH₃)₂ C₂₈H₄₀FN₅O₄ 530.32 530.2 63 F OH H CH₃ (CH₂)₂NHSO₂—CH₃C₂₇H₄₀FN₅O₅S 566.28 566.2 64 F OH H CH₃ (CH₂)₂N(CH₃)—C(O)OCH₃C₂₉H₄₅FN₅O₅ 560.33 560.2 65 F OH H CH₃ (CH₂)₂N(CH₃)—SO₂CH₃ C₂₈H₄₂FN₅O₅S580.30 580.2 66 F OH H CH₂CH₂OCH₃ (CH₂)₂N(CH₃)—SO₂CH₃ C₃₀H₄₆FN₅O₆S624.33 624.2 67 F OH H CH₂CH₂OH (CH₂)₂SO₂CH₃ C₂₈H₄₁FN₄O₆S 581.28 581.2(a) OC(O)N(CH₃)₂ (b) OC(O)NHCH₃

TABLE XXVII

[M + Molecular [M + H]⁺ H]⁺ No. R¹ Z Formula calcd found 1 H (CH₂)₂CH₂OHC₂₇H₃₈N₄O₄ 483.30 483.2 2 H (CH₂)₂OCH₃ C₂₈H₄₀N₄O₄ 497.31 497.3 3 H(CH₂)₂-2-pyrrolyl C₃₁H₄₁N₅O₃ 532.33 532.3 4 H CH₂-3-pyridinyl C₃₁H₃₉N₅O₃530.32 530.3 5 H (CH₂)₂NHC(O)OCH₃ C₂₉H₄₁N₅O₅ 540.32 540.3 6 H(CH₂)₂OC(O)N(CH₃)₂ C₃₀H₄₃N₅O₅ 554.34 554.3 7 H (CH₂)₂C(O)NHC₂H₅C₃₀H₄₃N₅O₄ 538.34 538.3 8 H (CH₂)₃C(O)N(CH₃)₂ C₃₁H₄₅N₅O₄ 552.36 552.3 9H (CH₂)₂NHSO₂CH₃ C₂₈H₄₁N₅O₅S 560.29 560.3 10 H (CH₂)₂N(CH₃)SO₂CH₃C₂₉H₄₃N₅O₅S 574.31 574.3 11 H (CH₂)₃SO₂N(CH₃)₂ C₃₀H₄₅N₅O₅S 588.32 588.312 H CH₂CF₃ C₂₇H₃₅F₃N₄O₃ 521.28 521.2 13 H (CH₂)₂C(O)NH₂ C₂₈H₃₉N₅O₄510.31 510.2 14 H CH₂CN C₂₇H₃₅N₅O₃ 478.28 478.2 15 H CH₂C(O)NH₂C₂₇H₃₇N₅O₄ 496.29 496.2 16 H CH₂C(O)N(CH₃)₂ C₂₉H₄₁N₅O₄ 524.33 524.2

TABLE XXVIII

Molecular [M + H]⁺ [M + H]⁺ No. R¹ R³ R⁴ W Formula calcd found 1 H OH H2-CH₂NHSO₂CH₃ C₂₉H₄₃N₅O₆S 590.30 590.2 2 H OH H 3-CH₂N(CH₃)SO₂CH₃C₃₀H₄₅N₅O₆S 604.32 604.2 3 H OH CH₃ 2-CH₂NHSO₂CH₃ C₃₀H₄₅N₅O₆S 604.32604.2 4 H OH H 3-CH₂OH C₂₈H₄₀N₄O₅ 513.31 513.2 5 H OCH₃ H 3-CH₂OHC₂₉H₄₂N₄O₅ 527.33 527.2 6 H OC(O)N(CH₃)₂ H 3-CH₂N(CH₃)SO₂CH₃ C₃₃H₅₀N₆O₇S675.36 675.4 7 H OC(O)N(CH₃)₂ H 3-CH₂OH C₃₁H₄₅N₅O₆ 584.35 584.2

Example 16 Radioligand Binding Assay on 5-HT_(4(c)) Human Receptors

a. Membrane Preparation 5-HT_(4(c))

HEK-293 (human embryonic kidney) cells stably-transfected with human5-HT_(4(c)) receptor cDNA (Bmax=˜6.0 pmol/mg protein, as determinedusing [³H]-GR113808 membrane radioligand binding assay) were grown inT-225 flasks in Dulbecco's Modified Eagles Medium (DMEM) containing4,500 mg/L D-glucose and pyridoxine hydrochloride (GIBCO-InvitrogenCorp., Carlsbad Calif.: Cat #11965) supplemented with 10% fetal bovineserum (FBS) (GIBCO-Invitrogen Corp.: Cat #10437), 2 mM L-glutamine and(100 units) penicillin-(100 μg) streptomycin/ml (GIBCO-Invitrogen Corp.:Cat #15140) in a 5% CO₂, humidified incubator at 37° C. Cells were grownunder continuous selection pressure by the addition of 800 μg/mLgeneticin (GIBCO-Invitrogen Corp.: Cat #10131) to the medium.

Cells were grown to roughly 60-80% confluency (<35 subculture passages).At 20-22 hours prior to harvesting, cells were washed twice and fed withserum-free DMEM. All steps of the membrane preparation were performed onice. The cell monolayer was lifted by gentle mechanical agitation andtrituration with a 25 mL pipette. Cells were collected by centrifugationat 1000 rpm (5 min).

For the membrane preparation, cell pellets were resuspended in ice-cold50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), pH7.4 (membrane preparation buffer) (40 mL/total cell yield from 30-40T225 flasks) and homogenized using a polytron disrupter (setting 19,2×10 s) on ice. The resultant homogenates were centrifuged at 1200 g for5 min at 4° C. The pellet was discarded and the supernatant centrifugedat 40,000 g (20 min). The pellet was washed once by resuspension withmembrane preparation buffer and centrifugation at 40,000 g (20 min). Thefinal pellet was resuspended in 50 mM HEPES, pH 7.4 (assay buffer)(equivalent 1 T225 flask/1 mL). Protein concentration of the membranesuspension was determined by the method of Bradford (Bradford, 1976).Membranes were stored frozen in aliquots at −80° C.

b. Radioligand Binding Assays

Radioligand binding assays were performed in 1.1 mL 96-deep wellpolypropylene assay plates (Axygen) in a total assay volume of 400 μLcontaining 2 μg membrane protein in 50 mM HEPES pH 7.4, containing0.025% bovine serum albumin (BSA). Saturation binding studies fordetermination of K_(d) values of the radioligand were performed using[³H]-GR113808 (Amersham Inc., Bucks, UK: Cat #TRK944; specific activity˜82 Ci/mmol) at 8-12 different concentrations ranging from 0.001 nM−5.0nM. Displacement assays for determination of pK_(i) values of compoundswere performed with [³H]-GR113808 at 0.15 nM and eleven differentconcentrations of compound ranging from 10 pM−100 μM.

Test compounds were received as 10 mM stock solutions in DMSO anddiluted to 400 μM into 50 mM HEPES pH 7.4 at 25° C., containing 0.1%BSA, and serial dilutions (1:5) then made in the same buffer.Non-specific binding was determined in the presence of 1 μM unlabeledGR113808. Assays were incubated for 60 min at room temperature, and thenthe binding reactions were terminated by rapid filtration over 96-wellGF/B glass fiber filter plates (Packard BioScience Co., Meriden, Conn.)presoaked in 0.3% polyethyleneimine. Filter plates were washed threetimes with filtration buffer (ice-cold 50 mM HEPES, pH7.4) to removeunbound radioactivity. Plates were dried, 35 μL Microscint-20 liquidscintillation fluid (Packard BioScience Co., Meriden, Conn.) was addedto each well and plates were counted in a Packard Topcount liquidscintillation counter (Packard BioScience Co., Meriden, Conn.).

Binding data were analyzed by nonlinear regression analysis with theGraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the 3-parameter model for one-site competition. The BOTTOM(curve minimum) was fixed to the value for nonspecific binding, asdetermined in the presence of 1 μM GR113808. K_(i) values for testcompounds were calculated, in Prism, from the best-fit IC₅₀ values, andthe K_(d) value of the radioligand, using the Cheng-Prusoff equation(Cheng and Prusoff, Biochemical Pharmacology, 1973, 22, 3099-108):K_(i)=IC₅₀/(1+[L]/K_(d)) where [L]=concentration [³H]-GR113808. Resultsare expressed as the negative decadic logarithm of the K_(i) values,pK_(i).

Test compounds having a higher pK_(i) value in this assay have a higherbinding affinity for the 5-HT₄ receptor. The compounds of the inventionwhich were tested in this assay had a pK_(i) value ranging from about 6to about 9.

Example 17 Radioligand Binding Assay on 5-HT_(3A) Human ReceptorsDetermination of Receptor Subtype Selectivity

a. Membrane Preparation 5-HT_(3A)

HEK-293 (human embryonic kidney) cells stably-transfected with human5-HT_(3A) receptor cDNA were obtained from Dr. Michael Bruess(University of Bonn, GDR) (Bmax=˜9.0 pmol/mg protein, as determinedusing [³H]-GR65630 membrane radioligand binding assay). Cells were grownin T-225 flasks or cell factories in 50% Dulbecco's Modified EaglesMedium (DMEM) (GIBCO-Invitrogen Corp., Carlsbad, Calif.: Cat #11965) and50% Ham's F12 (GIBCO-Invitrogen Corp.: Cat #11765) supplemented with 10%heat inactivated fetal bovine serum (FBS) (Hyclone, Logan, Utah: Cat#SH30070.03) and (50 units) penicillin-(50 μg) streptomycin/ml(GIBCO-Invitrogen Corp.: Cat #15140) in a 5% CO₂, humidified incubatorat 37° C.

Cells were grown to roughly 70-80% confluency (<35 subculture passages).All steps of the membrane preparation were performed on ice. To harvestthe cells, the media was aspirated and cells were rinsed with Ca²⁺,Mg²⁺-free Dulbecco's phosphate buffered saline (dPBS). The cellmonolayer was lifted by gentle mechanical agitation. Cells werecollected by centrifugation at 1000 rpm (5 min). Subsequent steps of themembrane preparation followed the protocol described above for themembranes expressing 5-HT_(4(c)) receptors.

b. Radioligand Binding Assays

Radioligand binding assays were performed in 96-well polypropylene assayplates in a total assay volume of 200 μL containing 1.5-2 μg membraneprotein in 50 mM HEPES pH 7.4, containing 0.025% BSA assay buffer.Saturation binding studies for determination of K_(d) values of theradioligand were performed using [³H]-GR65630 (PerkinElmer Life SciencesInc., Boston, Mass.: Cat #NET1011, specific activity ˜85 Ci/mmol) attwelve different concentrations ranging from 0.005 nM to 20 nM.Displacement assays for determination of pK_(i) values of compounds wereperformed with [³H]-GR65630 at 0.50 μM and eleven differentconcentrations of compound ranging from 10 μM to 100 μM. Compounds werereceived as 10 mM stock solutions in DMSO (see section 3.1), diluted to400 μM into 50 mM HEPES pH 7.4 at 25° C., containing 0.1% BSA, andserial (1:5) dilutions then made in the same buffer. Non-specificbinding was determined in the presence of 10 μM unlabeled MDL72222.Assays were incubated for 60 min at room temperature, then the bindingreactions were terminated by rapid filtration over 96-well GF/B glassfiber filter plates (Packard BioScience Co., Meriden, Conn.) presoakedin 0.3% polyethyleneimine. Filter plates were washed three times withfiltration buffer (ice-cold 50 mM HEPES, pH7.4) to remove unboundradioactivity. Plates were dried, 35 μL Microscint-20 liquidscintillation fluid (Packard BioScience Co., Meriden, Conn.) was addedto each well and plates were counted in a Packard Topcount liquidscintillation counter (Packard BioScience Co., Meriden, Conn.).

Binding data were analyzed using the non-linear regression proceduredescribed above to determine K_(i) values. The BOTTOM (curve minimum)was fixed to the value for nonspecific binding, as determined in thepresence of 10 μM MDL72222. The quantity [L] in the Cheng-Prusoffequation was defined as the concentration [³H]-GR65630.

Selectivity for the 5-HT₄ receptor subtype with respect to the 5-HT₃receptor subtype was calculated as the ratioK_(i)(5-HT_(3A))/K_(i)(5-HT_(4(c))). The compounds of the inventionwhich were tested in this assay had a 5-HT₄/5-HT₃ receptor subtypeselectivity ranging from about 10 to about 8000.

Example 18 Whole-cell cAMP Accumulation Flashplate Assay with HEK-293Cells Expressing Human 5-HT_(4(c)) Receptors

In this assay, the functional potency of a test compound was determinedby measuring the amount of cyclic AMP produced when HEK-293 cellsexpressing 5-HT₄ receptors were contacted with different concentrationsof test compound.

a. Cell Culture

HEK-293 (human embryonic kidney) cells stably-transfected with clonedhuman 5-HT_(4(c)) receptor cDNA were prepared expressing the receptor attwo different densities: (1) at a density of about 0.5-0.6 pmol/mgprotein, as determined using a [³H]-GR113808 membrane radioligandbinding assay, and (2) at a density of about 6.0 μmol/mg protein. Thecells were grown in T-225 flasks in Dulbecco's Modified Eagles Medium(DMEM) containing 4,500 mg/L D-glucose (GIBCO-Invitrogen Corp.: Cat#11965) supplemented with 10% fetal bovine serum (FBS) (GIBCO-InvitrogenCorp.: Cat #10437) and (100 units) penicillin-(100 μg) streptomycin/ml(GIBCO-Invitrogen Corp.: Cat #15140) in a 5% CO₂, humidified incubatorat 37° C. Cells were grown under continuous selection pressure by theaddition of geneticin (800 μg/mL: GIBCO-Invitrogen Corp.: Cat #10131) tothe medium.

b. Cell Preparation

Cells were grown to roughly 60-80% confluency. Twenty to twenty-twohours prior to assay, cells were washed twice, and fed, with serum-freeDMEM containing 4,500 mg/L D-glucose (GIBCO-Invitrogen Corp.: Cat#11965). To harvest the cells, the media was aspirated and 10 mL Versene(GIBCO-Invitrogen Corp.: Cat #15040) was added to each T-225 flask.Cells were incubated for 5 min at RT and then dislodged from the flaskby mechanical agitation. The cell suspension was transferred to acentrifuge tube containing an equal volume of pre-warmed (37° C.) dPBSand centrifuged for 5 min at 1000 rpm. The supernatant was discarded andthe pellet was re-suspended in pre-warmed (37° C.) stimulation buffer(10 mL equivalent per 2-3 T-225 flasks). This time was noted and markedas time zero. The cells were counted with a Coulter counter (count above8 μm, flask yield was 1-2×10⁷ cells/flask). Cells were resuspended at aconcentration of 5×10⁵ cells/ml in pre-warmed (37° C.) stimulationbuffer (as provided in the flashplate kit) and preincubated at 37° C.for 10 min.

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP(SMP004B, PerkinElmer Life Sciences Inc., Boston, Mass.), according tothe manufacturer's instructions.

Cells were grown and prepared as described above. Final cellconcentrations in the assay were 25×10³ cells/well and the final assayvolume was 100 μL. Test compounds were received as 10 mM stock solutionsin DMSO, diluted to 400 μM into 50 mM HEPES pH 7.4 at 25° C., containing0.1% BSA, and serial (1:5) dilutions then made in the same buffer.Cyclic AMP accumulation assays were performed with 11 differentconcentrations of compound ranging from 10 pM to 100 μM (final assayconcentrations). A 5-HT concentration-response curve (10 μM to 100 μM)was included on every plate. The cells were incubated, with shaking, at37° C. for 15 min and the reaction terminated by addition of 100 μl ofice-cold detection buffer (as provided in the flashplate kit) to eachwell. The plates were sealed and incubated at 4° C. overnight. Boundradioactivity was quantified by scintillation proximity spectroscopyusing the Topcount (Packard BioScience Co., Meriden, Conn.).

The amount of cAMP produced per mL of reaction was extrapolated from thecAMP standard curve, according to the instructions provided in themanufacturer's user manual. Data were analyzed by nonlinear regressionanalysis with the GraphPad Prism Software package using the 3-parametersigmoidal dose-response model (slope constrained to unity). Potency dataare reported as pEC₅₀ values, the negative decadic logarithm of the EC₅₀value, where EC₅₀ is the effective concentration for a 50% maximalresponse.

Test compounds exhibiting a higher pEC₅₀ value in this assay have ahigher potency for agonizing the 5-HT₄ receptor. The compounds of theinvention which were tested in this assay, for example, in the cell line(1) having a density of about 0.5-0.6 μmol/mg protein, had a pEC₅₀ valueranging from about 6 to about 9.

Example 19 In vitro Voltage Clamp Assay of Inhibition of Potassium IonCurrent in Whole Cells Expressing the hERG Cardiac Potassium Channel

CHO-K1 cells stably transfected with hERG cDNA were obtained from GailRobertson at the University of Wisconsin. Cells were held in cryogenicstorage until needed. Cells were expanded and passaged in Dulbecco'sModified Eagles Medium/F12 supplemented with 10% fetal bovine serum and200 μg/mL geneticin. Cells were seeded onto poly-D-lysine (100 μg/mL)coated glass coverslips, in 35 mm² dishes (containing 2 mL medium) at adensity that enabled isolated cells to be selected for whole cellvoltage-clamp studies. The dishes were maintained in a humidified, 5%CO₂ environment at 37° C.

Extracellular solution was prepared at least every 7 days and stored at4° C. when not in use. The extracellular solution contained (mM): NaCl(137), KCl (4), CaCl₂ (1.8), MgCl₂ (1), Glucose (10),4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) (10), pH 7.4with NaOH. The extracellular solution, in the absence or presence oftest compound, was contained in reservoirs, from which it flowed intothe recording chamber at approximately 0.5 mL/min. The intracellularsolution was prepared, aliquoted and stored at −20° C. until the day ofuse. The intracellular solution contained (mM): KCl (130), MgCl₂ (1),ethylene glycol-bis(beta-aminoethyl ether) N,N,N′,N′-tetra acetic acidsalt (EGTA) (5), MgATP (5),4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) (10), pH 7.2with KOH. All experiments were performed at room temperature (20-22°C.).

The coverslips on which the cells were seeded were transferred to arecording chamber and perfused continuously. Gigaohm seals were formedbetween the cell and the patch electrode. Once a stable patch wasachieved, recording commenced in the voltage clamp mode, with theinitial holding potential at −80 mV. After a stable whole-cell currentwas achieved, the cells were exposed to test compound. The standardvoltage protocol was: step from the holding potential of −80 mV to +20mV for 4.8 sec, repolarize to −50 mV for 5 sec and then return to theoriginal holding potential (−80 mV). This voltage protocol was run onceevery 15 sec (0.067 Hz). Peak current amplitudes during therepolarization phase were determined using pClamp software. Testcompounds at a concentration of 3 μM were perfused over the cells for 5minutes, followed by a 5-minute washout period in the absence ofcompound. Finally a positive control (cisapride, 20 nM) was added to theperfusate to test the function of the cell. The step from −80 mV to +20mV activates the hERG channel, resulting in an outward current. The stepback to −50 mV results in an outward tail current, as the channelrecovers from inactivation and deactivates.

Peak current amplitudes during the repolarization phase were determinedusing pCLAMP software. The control and test article data were exportedto Origin® (OriginLab Corp., Northampton Mass.) where the individualcurrent amplitudes were normalized to the initial current amplitude inthe absence of compound. The normalized current means and standarderrors for each condition were calculated and plotted versus the timecourse of the experiment.

Comparisons were made between the observed K⁺ current inhibitions afterthe five-minute exposure to either the test article or vehicle control(usually 0.3% DMSO). Statistical comparisons between experimental groupswere performed using a two-population, independent t-test (MicrocalOrigin v. 6.0). Differences were considered significant at p<0.05.

The smaller the percentage inhibition of the potassium ion current inthis assay, the smaller the potential for test compounds to change thepattern of cardiac repolarization when used as therapeutic agents. Forexample, the compounds of Examples 1-14, which were tested in this assayat a concentration of 3 μM, exhibited an inhibition of the potassium ioncurrent of less than about 25%, typically, less than about 15%.

Example 20 Pharmacokinetic Study in the Rat

Aqueous solution formulations of test compounds were prepared in 0.1%lactic acid at a pH of between about 5 and about 6. Male Sprague-Dawleyrats (CD strain, Charles River Laboratories, Wilmington, Mass.) weredosed with test compounds via intravenous administration (IV) at a doseof 2.5 mg/kg or by oral gavage (PO) at a dose of 5 mg/kg. The dosingvolume was 1 mL/kg for IV and 2 mg/kg for PO administration. Serialblood samples were collected from animals pre-dose, and at 2 (IV only),5, 15, and 30 min, and at 1, 2, 4, 8, and 24 hours post-dose.Concentrations of test compounds in blood plasma were determined byliquid chromatography-mass spectrometry analysis (LC-MS/MS) (MDS SCIEX,API 4000, Applied Biosystems, Foster City, Calif.) with a lower limit ofquantitation of 1 ng/mL.

Standard pharmacokinetic parameters were assessed by non-compartmentalanalysis (Model 201 for IV and Model 200 for PO) using WinNonlin(Version 4.0.1, Pharsight, Mountain View, Calif.). The maximum in thecurve of test compound concentration in blood plasma vs. time is denotedC_(max). The area under the concentration vs. time curve from the timeof dosing to the last measurable concentration (AUC(0−t)) was calculatedby the linear trapezoidal rule. Oral bioavailability (F(%)), i.e. thedose-normalized ratio of AUC(0−t) for PO administration to AUC(0−t) forIV administration, was calculated as:F(%)=AUC _(PO) /AUC _(IV)×Dose_(IV)/Dose_(PO)×100%

Test compounds which exhibit larger values of the parameters C_(max),AUC(0−t), and F(%) in this assay are expected to have greaterbioavailability when administered orally. For example, the compounds ofExamples 1-14 were tested in this assay and had C_(max) values typicallyranging from about 0.05 to about 0.4 μg/mL and AUC(0−t) values typicallyranging from about 0.15 to about 0.9 μg·hr/mL.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. Additionally, all publications, patents, andpatent documents cited hereinabove are incorporated by reference hereinin full, as though individually incorporated by reference.

1. A pharmaceutical composition comprising a pharmaceutically-acceptablecarrier and a compound of formula (I):

wherein: R¹ is hydrogen, halo, hydroxy, C₁₋₄alkyl, or C₁₋₄alkoxy; R² isC₃₋₄alkyl or C₃₋₆cycloalkyl; R³ is hydroxy, C₁₋₃alkoxy,hydroxy-substituted C₁₋₄alkyl, or —OC(O)NR^(a)R^(b); R⁴ is hydrogen orC₁₋₄alkyl; X is selected from —N(R⁸)C(O)R⁹, —N(R⁸)S(O)₂R¹⁰, —S(R¹¹)O₂,—N(R⁸)C(O)OR¹², —N(R⁸)C(O)NR¹³R¹⁴, —N(R⁸)SO₂NR¹³R¹⁴, —C(O)NR¹³R¹⁴,—OC(O)NR¹³R¹⁴, —C(O)OR¹², —OR¹⁵, and cyano; R⁵ is hydrogen or C₁₋₄alkyl,wherein C₁₋₄alkyl is optionally substituted with hydroxy, C₁₋₃alkoxy, orcyano; R⁶ and R⁷ are independently selected from hydrogen, hydroxy,halo, and C₁₋₄alkyl, wherein C₁₋₄alkyl is optionally substituted withhydroxy or C₁₋₃alkoxy; R⁸ is hydrogen or C₁₋₄alkyl; or R⁵ and R⁸, R⁵ andR⁶, or R⁶ and R⁸ taken together form C₂₋₅alkylenyl, whereinC₂₋₅alkylenyl is optionally substituted with hydroxy, halo,hydroxy-substituted C₁₋₃alkyl, or C₁₋₃alkoxy; or R³ and R⁵ takentogether form —OCH₂CH₂—; or R⁵ and R⁶ taken together form—(CH₂)_(q)-Q-(CH₂)_(q), wherein Q is oxygen or sulfur and q isindependently 0, 1, or 2; R⁹ is hydrogen, furanyl, or C₁₋₄alkyl, whereinC₁₋₄alkyl is optionally substituted with hydroxy or with from 1 to 3halo; R¹⁰ is hydrogen or C₁₋₄alkyl, wherein C₁₋₄alkyl is optionallysubstituted with —SO₂R^(c), C₃₋₆cycloalkyl or with from 1 to 3 halo; orR⁸ and R¹⁰ taken together form C₃alkylenyl; R¹¹ is hydrogen, C₁₋₄alkyl,or —NR^(b)R^(c); or R⁵ and R¹¹ or R⁶ and R¹¹ taken together formC₂₋₅alkylenyl; R¹², R¹³, and R¹⁴ are independently hydrogen orC₁₋₄alkyl; R¹⁵ is hydrogen or C₁₋₄alkyl, wherein C₁₋₄alkyl is optionallysubstituted with hydroxy; R^(a), R^(b), and R^(c) are independentlyhydrogen or C₁₋₃alkyl; and n is 1, 2, 3, or 4; provided that when n is1, X forms a carbon-carbon bond with the carbon atom bearing thesubstituents R⁶ and R⁷; or a pharmaceutically-acceptable salt orstereoisomer thereof.
 2. The pharmaceutical composition of claim 1wherein: R⁵ is hydrogen or C₁₋₄alkyl, wherein C₁₋₄alkyl is optionallysubstituted with hydroxy, C₁₋₃alkoxy, or cyano; R⁶ and R⁷ areindependently selected from hydrogen, hydroxy, halo, and C₁₋₄alkyl,wherein C₁₋₄alkyl is optionally substituted with hydroxy or C₁₋₃alkoxy;R⁸ is hydrogen or C₁₋₄alkyl; R¹⁰ is hydrogen or C₁₋₄alkyl, whereinC₁₋₄alkyl is optionally substituted with —SO₂R^(c), C₃₋₆cycloalkyl orwith from 1 to 3 halo; R¹¹ is hydrogen, C₁₋₄alkyl, or —NR^(b)R^(c); andR¹⁵ is hydrogen or C₁₋₄alkyl, wherein C₁₋₄alkyl is optionallysubstituted with hydroxy.
 3. The pharmaceutical composition of claim 1wherein R² is C₃₋₄alkyl.
 4. The pharmaceutical composition of claim 3wherein n is 2 or
 3. 5. The pharmaceutical composition of claim 1wherein the compound of formula (I) is a compound of formula (II):

wherein: R¹ is hydrogen, halo, or C₁₋₃alkyl; R² is C₃₋₄alkyl; R³ ishydroxy, C₁₋₃alkoxy, hydroxy-substituted C₁₋₂alkyl, or—OC(O)NR^(a)R^(b); R⁵ is hydrogen, C₁₋₃alkyl, or C₁₋₃alkyl substitutedat the terminal position with hydroxy or cyano; R⁶ is hydrogen; X isselected from —N(R⁸)C(O)R⁹; —N(R⁸)S(O)₂R¹⁰, —S(R¹¹)O₂,—N(R⁸)C(O)NR¹³R¹⁴, —C(O)NR¹³R¹⁴, —OC(O)NR¹³R¹⁴, —OR¹⁵, and cyano; R⁸ ishydrogen or C₁₋₃alkyl; R⁹ is hydrogen or C₁₋₃alkyl; R¹⁰ is hydrogen orC₁₋₃alkyl, wherein C₁₋₃alkyl is optionally substituted with —SO₂R^(c)wherein R^(c) is C₁₋₃alkyl; R¹³, R¹⁴, and R¹⁵ are independently hydrogenor C₁₋₃alkyl; or R⁵ and R⁸, R⁵ and R⁶, or R⁵ and R¹¹ taken together formC₂alkylenyl; or a pharmaceutically-acceptable salt or stereoisomerthereof.
 6. The pharmaceutical composition of claim 5 wherein: R¹ ishydrogen; R² is C₃₋₄alkyl; R³ is hydroxy, methoxy, hydroxymethyl,—OC(O)N(H)CH₃, or —OC(O)N(CH₃)₂; R⁶ is hydrogen; X is selected from—N(R⁸)C(O)R⁹; —N(R⁸)S(O)₂R¹⁰, —S(R¹¹)O₂, and —N(R⁸)C(O)NR¹³R¹⁴; R⁵ andR⁸ taken together form C₂alkylenyl; R⁹ is hydrogen or C₁₋₃alkyl; R¹⁰ ishydrogen, C₁₋₃alkyl, or methanesulfonylmethyl; R⁵ and R¹¹ taken togetherform C₂alkylenyl; and R¹³ and R¹⁴ are independently hydrogen orC₁₋₃alkyl.
 7. The pharmaceutical composition of claim 1 wherein thecompound of formula (I) is a compound of formula (III):


8. The pharmaceutical composition of claim 1 wherein the compound offormula (I) is a compound of formula (IV):


9. The pharmaceutical composition of claim 1 wherein the compound offormula (I) is a compound of formula (V):


10. The pharmaceutical composition of claim 1 wherein the compound offormula (I) is a compound selected from:1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid((1S,3R,5R)-8-{2-hydroxy-3-[4-(propane-2-sulfonyl)piperazin-1-yl]propyl}-8-azabicyclo[3.2.1]oct-3-yl)-amide;1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(1,1-dioxo-1λ⁶-thiomorpholin-4-yl)-2-hydroxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(S)-2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[(R)-2-hydroxy-3-(4-methanesulfonylpiperazin-1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(4-methanesulfonylmethanesulfonylpiperazin-1-yl)-2-methoxypropyl]-8-azabicyclo[3.2.1]-oct-3-yl}amide;1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(4-acetylpiperazin-1-yl)-2-methoxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-(1,1-dioxo-1λ⁶-thiomorpholin-4-yl)-2-methoxypropyl]-8-azabicyclo[3.2.1]oct-3-yl}amide;methylcarbamic acid2-{(1S,3R,5R)-3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}-1-(4-methanesulfonylpiperazin-1-yl-methyl)ethylester; methylcarbamic acid1-(4-dimethylcarbamoylpiperazin-1-ylmethyl)-2-{(1S,3R,5R)-3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-8-aza-bicyclo[3.2.1]oct-8-yl}ethylester; methylcarbamic acid1-[3-(acetylmethylamino)pyrrolidin-1-ylmethyl]-2-{(1S,3R,5R)-3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-8-aza-bicyclo[3.2.1]oct-8-yl}ethylester; methylcarbamic acid1-(4-acetylpiperazin-1-ylmethyl)-2-{(1S,3R,5R)-3-[(1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}-ethylester; methylcarbamic acid(R)-2-{(1S,3R,5R)-3-[(1-isopropyl-2-oxo-1,2-dihydro-quinoline-3-carbonyl)amino]-8-azabicyclo[3.2.1]oct-8-yl}-1-(4-methanesulfonyl-piperazin-1-ylmethyl)ethylester; and 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic acid{(1S,3R,5R)-8-[3-hydroxy-2-(4-methanesulfonylpiperazin-1-ylmethyl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}-amide;and pharmaceutically-acceptable salts thereof.